Is glycine "sweet" to mice? Mouse strain differences in perception of glycine taste

Glycine is an amino acid tasting sweet to humans. In 2-bottle tests, C57BL/6ByJ (B6) mice strongly prefer glycine solutions, whereas 129P3/J (129) mice do not, suggesting that they differ in perception of glycine taste. We examined this question using the conditioned taste aversion (CTA) generalization technique. CTA was achieved by injecting LiCl after drinking glycine, and next its generalization to 10 taste solutions (glycine, sucrose, saccharin, D-tryptophan, L-tryptophan, L-alanine, L-proline, L-glutamine, NaCl, and HCl) was examined by video recording licking behavior. Both B6 and 129 mice generalized the aversion to sucrose, saccharin, L-alanine, and L-proline and did not generalize it to NaCl, HCl, and L-tryptophan. This indicates that both B6 and 129 mice perceive the sweetness (i.e., a sucrose-like taste) of glycine. Thus, the lack of a glycine preference by 129 mice cannot be explained by their inability to perceive its sweetness. Strain differences were observed for CTA generalization to 2 amino acids: 129 mice generalized aversion to L-glutamine but not D-tryptophan, whereas B6 mice generalized it to D-tryptophan but not L-glutamine. 129.B6-Tas1r3 congenic mice with 2 genotypes of the Tas1r3 locus (B6/129 heterozygotes and 129/129 homozygotes) did not differ in aversion generalization, suggesting that the differences between 129 and B6 strains are not attributed to the Tas1r3 allelic variants and that other, yet unknown, genes are involved in taste perception of amino acids.     

Need rods? Get glycine receptors and taurine

Taurine, a multifunctional amino acid prevalent in developing nervous tissues, regulates the number of rod photoreceptors in developing postnatal rodent retina. In this issue of Neuron, Young and Cepko show that taurine acts via GlyRalpha2 subunit-containing glycine receptors expressed by retinal progenitor cells at birth.     

PKCβ-dependent phosphorylation of the glycine transporter 1

The extracellular levels of the neurotransmitter glycine in the brain are tightly regulated by the glycine transporter 1 (GlyT1) and the clearance rate for glycine depends on its rate of transport and the levels of cell surface GlyT1. Over the years, it has been shown that PKC tightly regulates the activity of several neurotransmitter transporters. In the present work, by stably expressing three N-terminus GlyT1 isoforms in porcine aortic endothelial cells and assaying for [³²P]-orthophosphate metabolic labeling, we demonstrated that the isoforms GlyT1a, GlyT1b, and GlyT1c were constitutively phosphorylated, and that phosphorylation was dramatically enhanced, in a time dependent fashion, after PKC activation by phorbol ester. The phosphorylation was PKC-dependent, since pre-incubation of the cells with bisindolylmaleimide I, a selective PKC inhibitor, abolished the phorbol ester-induced phosphorylation. Blotting with specific anti-phospho-tyrosine antibodies did not yield any signal that could correspond to GlyT1 tyrosine phosphorylation, suggesting that the phosphorylation occurs at serine and/or threonine residues. In addition, a 23-40%-inhibition on V_max was obtained by incubation with phorbol ester without a significant change on the apparent Km value. Furthermore, pre-incubation of the cells with the selective PKCα/β inhibitor Gö6976 abolished the downregulation effect of phorbol ester on uptake and phosphorylation, whereas the selective PKCβ inhibitors (PKCβ inhibitor or LY333531) prevented the phosphorylation without affecting glycine uptake, defining a specific role of classical PKC on GlyT1 uptake and phosphorylation. Taken together, these data suggest that conventional PKCα/β regulates the uptake of glycine, whereas PKCβ is responsible for GlyT1 phosphorylation.     

Inward-facing glycine residues create sharp turns in β-barrel membrane proteins

The transmembrane region of outer-membrane proteins (OMPs) of Gram-negative bacteria are almost exclusively β-barrels composed of between 8 and 26 β-strands. To explore the relationship between β-barrel size and shape, we modeled and simulated engineered variants of the Escherichia coli protein OmpX with 8, 10, 12, 14, and 16 β-strands. We found that while smaller barrels maintained a roughly circular shape, the 16-stranded variant developed a flattened cross section. This flat cross section impeded its ability to conduct ions, in agreement with previous experimental observations. Flattening was determined to arise from the presence of inward-facing glycines at sharp turns in the β-barrel. An analysis of all simulations revealed that glycines, on average, make significantly smaller angles with residues on neighboring strands than all other amino acids, including alanine, and create sharp turns in β-barrel cross sections. This observation was generalized to 119 unique structurally resolved OMPs. We also found that the fraction of glycines in β-barrels decreases as the strand number increases, suggesting an evolutionary role for the addition or removal of glycine in OMP sequences.     

Asymmetric synthesis of (S)-α-(octyl)glycine via alkylation of Ni(II) complex of chiral glycine Schiff base

Over last decade, the use of Ni(II) complexes, derived from of glycine Schiff bases with chiral tridentate ligands, has emerge as a leading methodology for preparation of structurally diverse Tailor-Made Amino Acids, the key structural units in modern medicinal chemistry, and drug design. Here, we report asymmetric synthesis of derivatives of (S)-α-(octyl)glycine ((S)-2-aminodecanoic acid) and its N-Fmoc derivative via alkylation of chiral nucleophilic glycine equivalent with n-octyl bromide. Under the optimized conditions, the alkylation proceeds with excellent yield (98.1%) and diastereoselectivity (98.8% de). The observed stereochemical outcome and convenient reaction conditions bode well for application of this method for large-scale asymmetric synthesis of (S)-2-aminodecanoic acid and its derivatives.     

Polymerization rate difference of N-alkyl glycine NCAs: Steric hindrance or not?

Polypeptoids synthesized from N-substituted glycine N-carboxyanhydrides (NNCAs) are widely applied in biological fields. The effect of side groups in NNCA polymerizations is a key to develop novel polypeptoids with complex topologies and constituents. In this work, density functional theory (DFT) calculations are employed to investigate the propagation of a series of alkyl substituted NNCAs with solvation model. According to both computational and experimental results, carbonyl addition is confirmed as rate determining step and steric hindrance is recognized as the major factor of low reactivity in β-C branched NNCAs. However, in linear and γ-C branched case, aggregation of side groups instead of bulkiness is considered responsible for low polymerization rate.     

Two new reactive targets of 2,5-hexanedione in vitro – beta-alanine and glycine

Summary. In this study, we found that two amino acids reacted with 2,5-hexanedione to form new reaction products in vitro, respectively. In the reaction of beta-alanine and 2,5-hexanedione, a reaction product was obtained and analyses of obtained results showed it was 3-(2,5-dimethyl-1H-pyrrol-1-yl)propanoic acid; in the reaction of glycine and 2,5-hexanedione, a reaction product was also obtained and analyses showed it was (2,5-dimethyl-1H-pyrrol-1-yl)acetic acid. Two reaction products were found to be oxidized easily; in addition, the latter was more easily to be oxidized than the former in the air. Our discoveries demonstrated that reactions between amino acids and 2,5-hexanedione could exist possibly in vitro. At present, it is clear that 2,5-hexanedione causes either axon atrophy or swelling, but the underlying molecular mechanism is still unclear. Since both beta-alanine and glycine are considered as neurotransmitter in the central nervous system, the reaction products remain to be identified in vivo.     

Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy

In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 μm^?2, respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.     

Production of thermophilic α-amylase using immobilized transformed Escherichia coli by addition of glycine

Efficient production of thermophilic α-amylase from Bacillus stearothermophilus was investigated using recombinant Escherichia coli HB101/pH1301 immobilized with κ-carrageenan by the addition of glycine. The effects of glycine, the concentrations of κ-carrageenan and KCI on the production of the enzyme as well as the stability of plasmid pHI301 were studied. In the absence of glycine, the enzyme was localized in the periplasmic space of the recombinant E. coli cells and a small amount of the enzyme was liberated in the culture broth. Although the addition of glycine was very effective for release of α-amylase from the periplasm of E. coli entrapped in gel beads, a majority of the enzyme accumulated in the gel matrix. (In this paper, production of the enzyme from recombinant cells to an ambient is expressed by the term “release”, while diffusion-out from gel beads is referred to by the term “liberate”.) Concentrations of KCI and immobilizing support significantly affected on the liberation of α-amylase to the culture broth. Mutants which produced smaller amounts of the enzyme emerged during a successive culture of recombinant E. coli, even under selective pressure, and they predominated in the later period of the passages. The population of plasmid-lost segregants increased with cultivation time. The stability of pHI301 for the free cells was increased by the addition of 2% KCI, which is a hardening agent for carrageenan. Although the viability of cells and α-amylase activity in the beads decreased with cultivation time during the successive culture of the immobilized recombinant E. coli, the plasmid stability was increased successfully by immobilization. Efficient long-term production of α-amylase was attained by an iterative re-activation-liberation procedure using the immobilized recombinant cells. Although the viable cell number, plasmid stability and enzyme activity liberated in the glycine solution decreased at an early period in the cultivation cycles, the process attained steady state regardless of the addition of an antibiotic.     

Modulation of NMDA receptors by glycine — introduction to some basic aspects and recent developments

Summary Glycine is a co-agonist at NMDA receptors and it's presence is a prerequisite for channel activation by glutamate or NMDA. Physiological concentrations reduce one form of NMDA receptor-desensitization. Interactions between the glycine_B site and other domains of the NMDA receptor are complex and include the glutamate, Mg⁺ and polyamines sites. Glycine shows different affinities at various NMDA receptor subtypes probably via to allosteric interactions between NMDA2 subunits and the glycine recognition site on the NMDAR1 subunit. There is still some debate whether the glycine_B site is saturatedin vivo but it seems likely that this depends on regional differences in receptor subtype expression, local glycine or D-serene concentrations and the expression of specific glycine transporters.Glycine_B antagonists and partial agonists have been reported to have good therapeutic indices as neuroprotective agents against focal ischaemia and trauma, anti-epileptics, anxiolytics, anti-psychotomimetics and in models of chronic pain. They clearly lack two potentially serious side effects classically associated with NMDA receptor blockade, namely neurodegenerative changes in the cingulate/retrosplenial cortex and psychotomimetic-like effects. This improved therapeutic profile may be partially due to the ability of full glycine_B antagonists to reveal Gycne-sensitive desensitization and possibly also via functional and/or regional NMDA receptor subtype selectivity.     

Is glycine betaine a non-compatible solute in higher plants that do not accumulate it?

Rape (Brassica napus L. var. oleifera cv. Samourai) leaf discs treated in vitro in the presence of glycine betaine (GB) exhibited very high accumulation of GB, suggesting the operation of a specific uptake system. When further subjected to osmotic upshocks by transfer to PEG 6000 media, the typical osmo-induced proline response of the discs was strongly inhibited. The level of this inhibition was quantitatively related to the amount of GB loaded in the tissues. In contrast, the soluble sugar content increased in GB-treated discs. Surprisingly, viability tests (i.e. membrane stability and 2,3,5-triphenyltetrazolium chloride reduction) indicated a destabilizing effect of GB in these tissues. This is at variance with the relative compatibility of sucrose and proline. In addition, the protein content was lower in GB-treated discs. This could be related to an inhibitory effect on protein synthesis, as demonstrated by radiolabelling of polypeptides with [³⁵S] amino acids. This effect was particularly pronounced on Rubisco large sub-unit synthesis and was still apparent under non-stress conditions. The GB treatment was also followed by the induction or up-regulation of a set of polypeptides, not seen under stress conditions, while the synthesis of osmo-induced polypeptides was not affected by GB. These novel effects of GB lead us to discuss the reasons for its incompatibility in leaf tissues of a non-GB-accumulating species.     

Exogenously supplied glycine betaine in spinach and rapeseed leaf discs: compatibility or non-compatibility?

CMS, cell membrane stability
GB, glycine betaine
PEG, polyethylene glycol
TTC, 2,3,5-triphenyltetrazolium chloride

When leaf discs of spinach ( Spinacia oleracea cv. Junius) and rapeseed ( Brassica napus var. oleifera cv. Samourai) were incubated in the light in the presence of glycine betaine (GB), they accumulated GB at a very high level. In comparison with the spinach leaf explants, the uptake of GB by rapeseed tissues was restricted, probably by the destabilizing effects exerted by GB in this plant material. In contrast, the viability of spinach leaf discs, as assessed by their capacity to reduce 2,3,5-triphenyltetrazolium chloride (TTC), was not affected, suggesting that the GB taken up was compatible in the leaf tissues of the GB accumulator. In rapeseed leaf discs treated with GB, chlorophyll loss as well as significant changes in polyamine content were induced, leading to a dramatic increase of the putrescine/(spermidine + spermine) ratio. In contrast, this ratio remained constant in the GB treated spinach explants, suggesting that spinach has the capacity to stabilize polyamine metabolism in the presence of high amounts of GB. The treatment of spinach leaf discs with GB prior to application of osmotic or salt shocks provided protection from stress. A weak capacity to accumulate proline under stress conditions was partially suppressed. The protein content decreased while the free amino acid level increased independently of the presence of GB. It is concluded that GB behaves as a true compatible solute in spinach, which is a typical GB accumulator, and that GB is damaging when loaded into the leaf tissues of rapeseed, which do not normally accumulate GB.     

Delayed supplementation of glycine enhances extracellular secretion of the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli

The targeting of recombinant proteins for secretion to the culture medium of Escherichia coli presents significant advantages over cytoplasmic or periplasmic expression. However, a major barrier is inadequate secretion across two cell membranes. In the present study, we attempted to circumvent this secretion problem of the recombinant α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01. It was found that glycine could promote extracellular secretion of the recombinant α-CGTase for which one potential mechanism might be the increase in membrane permeability. However, further analysis indicated that glycine supplementation resulted in impaired cell growth, which adversely affected overall recombinant protein production. Significantly, delayed supplementation of glycine could control cell growth impairment exerted by glycine. As a result, if the supplementation of 1% glycine was optimally carried out at the middle of the exponential growth phase, the α-CGTase activity in the culture medium reached 28.5 U/ml at 44 h of culture, which was 11-fold higher than that of the culture in regular terrific broth medium and 1.2-fold higher than that of the culture supplemented with 1% glycine at the beginning of culture.     

Protonation equilibria of glycine, 1,10-phenanthroline and 2,2-bipyridyl in ethylene glycol–water mixtures

Abstract

The solute–solvent interactions of glycine, 1,10-phenanthroline and 2,2-bipyridyl have been studied in 0–60% v/v ethylene glycol–water media by a pH metric method. The protonation constants were estimated with the computer program MINIQUAD75. Selection of the best fit chemical model of the protonation equilibria is based on the standard deviation in protonation constants and residual analysis using a sum of squares of residuals in all mass-balance equations. The observed linear variation of protonation constants with the inverse of dielectric constant of the solvent mixture can be attributed to the dominance of the electrostatic forces. The distribution of species, protonation equilibria and effects of influential parameters on the protonation constants are also presented.     

Amino acid accumulation in frog muscle: I. Steady-state glycine accumulation at o °C

Glycine is not significantly metabolized by frog muscle maintained at o °C in vitro. Nevertheless, in this preparation steady-state levels of [¹⁴C]glycine as high as 20 times the external concentration are attained after 3–6 days at o °C. The concentration gradient at the steady state depends on the external concentration, being highest at low external concentrations (approx. 0.1 mM) and reversed at external concentrations above 10 mM.A plot of the steady-state cellular levels of glycine vs the external concentration reveal linear and saturable components. The linear fraction has an average distribution ratio of 0.54 indicating that glycine is partially excluded from the muscle water at this temperature.Efflux of labeled glycine at o °C from previously loaded frog muscle follows first-order kinetics. The rate constant increases with increasing concentrations of glycine in the external medium (efflux facilitation).The steady-state results are shown to be consistent with an adsorption model for amino acid accumulation as well as a model in which amino acid enters the cell via a carrier and exits via a bidirectional leakage pathway. A model in which efflux proceeds through the carrier does not fit the data. This indicates that an alternative to exchange diffusion is needed to explain the observed efflux facilitation.     

Natural fermentation of soybean (glycine max) with added salt for ‘soy-daddawa’ production

Microbial growth, pH and titratable acidity decreased with increasing NaCl concentration during the fermentation of soybeans. NaCl at 1% (w/w) in the fermenting mash improved the organoleptic quality of soy-daddawa.The author is with the Department of Microbiology, Obafemi Awolowo University, lle-lfe, Nigeria.     

The role of β-dimethylsulphoniopropionate,glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis

The tertiary sulphonium compound, -dimethylsulphoniopropionate (DMSP) and the quaternary ammonium compounds glycine betaine and homarine are important osmotica in Platymonas subcordiformis cells. Following hypersaline stresses the compounds were accumulated after a lag period of 3 h and equilibrium concentrations were reached 6 h later. In contrast to these organic solutes, mannitol was synthesised immediately and equilibrium concentrations were reached within 90 min. Hyposaline stresses induced losses of the organic solutes from the cells. The ions K⁺, Na⁺, Cl^- and the above organic solutes can account for the osmotic balance of the cells.Abbreviations DMSP -dimethylsulphoniopropionate - _i intracellular osmolality - _o extracellular osmolality     

The trafficking proteins Vacuolar Protein Sorting 35 and Neurobeachin interact with the glycine receptor β-subunit

Inhibitory glycine receptors (GlyRs) are densely packed in the postsynaptic membrane due to a high-affinity interaction of their β-subunits with the scaffolding protein gephyrin. Here, we used an affinity-based proteomic approach to identify the trafficking proteins Vacuolar Protein Sorting 35 (Vps35) and Neurobeachin (Nbea) as novel GlyR β-subunit (GlyRβ) interacting proteins in rat brain. Recombinant Vps35 and a central fragment of Nbea bound to the large intracellular loop of GlyRβ in glutathione-S-transferase pull-downs; in addition, Vps35 displayed binding to gephyrin. Immunocytochemical staining of spinal cord sections revealed Nbea immunoreactivity apposed to and colocalizing with marker proteins of inhibitory synapses. Our data are consistent with roles of Vps35 and Nbea in the retrieval and post-Golgi trafficking of synaptic GlyRs and possibly other neurotransmitter receptors.     

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

Background

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.

Methods

Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.

Results

Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.

Conclusions

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.

General significance

This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.