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Glycine derivatives of imidazolones as potential ligands of glycine binding site of NMDA receptors. Part 1.

The series of glycine derivatives of diphenyl or (un)substituted arylidene imidazolones was designed and obtained as potential ligands of the glycine binding site of NMDA receptors. The compounds were evaluated in vitro for their affinity to the glycine binding site of NMDA receptors using as radioligand [3H]-L-689,560. Their anticonvulsant activity was estimated in vivo in maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scMet) tests. To explain the structure, pharmacological activity results Multiple Linear Regression Analysis was used.

Animals↗

Evidence for indirect utilization of glycine for production of N-bases of glycerophospholipids and sphingolipids in mouse neuroblastoma cells by using 15N-labeled glycine.

After mouse neuroblastoma cells were cultured with [15N]glycine in Dulbecco's modified Eagle's medium for total four days, they gave significant amounts of 15N-atom% excess in phosphatidylserine, phosphatidylethanolamine, ethanolamine plasmalogen, phosphatidylcholine, sphingomyelin and ceramide. These results were compared with those of [15N]choline, [15N]ethanolamine and [15N]DL-serine used in place of [15N]glycine for the experiments. It was suggested that [15N]glycine is taken into the cells and metabolically converted to [15N]L-serine, which is well known to be used for the production of polar head groups of glycerophospholipids and sphingosine.

Animals↗

Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate.

The possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [3H]L-glutamate [( 3H]GLU) and [3H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [3H]Glycine always labelled a single population of sites; densities of binding sites (Bmax) in cortical, cerebellar and "granule" membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (Kd) in the same three preparations were 0.13, 0.31 and 1.9 microM, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [3H]GLU was saturable and to a single population of sites: Kd 0.5-0.9 microM and Bmax 3.2-3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [3H]GLU was always L-aspartate greater than L-cysteate greater than L-cysteinesulphinate greater than L-serine-O-sulphate greater than ibotenate greater than L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [3H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Effect of glycine and glycine receptor antagonists on NMDA-induced brain injury.

In postnatal day 7 rats, a unilateral intrastriatal injection of 12.5 nmol of N-methyl-D-aspartate (NMDA) reproducibly injures the ipsilateral striatum, adjacent hippocampus and overlying cortex. The severity of injury can be quantified by comparing cerebral hemisphere weights in animals sacrificed 5 days after the injection. Co-injection of NMDA and the glycine receptor antagonists kynurenic acid (KYN) or 7-chlorokynurenic acid (7-CKA) reduced the severity of NMDA-induced damage in a dose-dependent fashion. One hundred nmol of KYN with 12.5 nmol of NMDA reduced average % damage from 19.3 +/- 0.9% (n = 9) to 2.3 +/- 0.5% (n = 6), P less than 0.001, ANOVA. Co-injection of 40 nmol of 7-CKA with 12.5 nmol of NMDA (n = 6) reduced average % damage from 17.1 +/- 1.6% (n = 15) to 3.0 +/- 0.6%, P less than 0.001, ANOVA. Concurrent injection of 1000 nmol glycine with 5 nmol NMDA did not increase the extent of NMDA-induced damage. Our results demonstrate that glycine receptor antagonists attenuate NMDA-induced brain injury in vivo.

Animals↗

Metabolism of 1-14C glyoxylate, 1-14C glycollate, 1-14C glycine and 2-14C glycine by homogenates of kidney and liver tissue from hyperoxaluric and control subjects.

1. The metabolism of [1-(14)C]glyoxylate to carbon dioxide, glycine, oxalate, serine, formate and glycollate was investigated in hyperoxaluric and control subjects' kidney and liver tissue in vitro. 2. Only glycine and carbon dioxide became significantly labelled with (14)C, and this was less in the hyperoxaluric patients' kidney tissue than in the control tissue. 3. Liver did not show this difference. 4. The metabolism of [1-(14)C]glycollate was also studied in the liver tissue; glyoxylate formation was demonstrated and the formation of (14)CO(2) from this substrate was likewise unimpaired in the hyperoxaluric patients' liver tissue in these experiments. 5. Glycine was not metabolized by human kidney, liver or blood cells under the conditions used. 6. These observations show that glyoxylate metabolism by the kidney is impaired in primary hyperoxaluria.

Adult↗

Two cysteine substitutions in procollagen I: a glycine replacement near the N-terminus of alpha 1(I) chain causes lethal osteogenesis imperfecta and a glycine replacement in the alpha 2(I) chain markedly destabilizes the triple helix.

Cultured skin fibroblasts were examined from two probands with type II (lethal) osteogenesis imperfecta. One proband had a single base mutation which converted the glycine codon at position alpha 1-244 in the alpha 1(I) chain of procollagen I into a cysteine codon whereas the other had a similar mutation that converted the glycine codon at position alpha 2-787 of the alpha 2(I) chain into a cysteine codon. Both mutations produced post-translational overmodification of procollagen I. The Cys alpha 1-244 mutation, however, had a minimal effect on the thermal stability or secretion of the protein whereas the Cys alpha 2-787 mutation markedly decreased the thermal stability and, apparently as a result, essentially none of the mutated protein was secreted. The results provide clear exceptions to two previous generalizations about the position-specificity of glycine substitutions in procollagen I.

Base Sequence↗

[Changes of polyamines level in Glycine soja and Glycine max seedlings under NaCl stress].

With internationally common-used Glycine max (the salt-tolerant Lee68) and Glycine soja (the salt-sensitive N23232) as reference, this paper studied the polyamines (PAs) contents and polyamine oxidase (PAO) activities in the highly salt-tolerant BB52 (Glycine soja) seedlings, which showed that under 150mmol x L(-1) NaCl stress for 2d, the decrease of Put and Spd contents was more significant, but that of Spd content was less significant in roots of BB52 than in those of Lee68 and N23232. For leaves, the decrease of Put and increase of Spd contents were markedly observed in BB52. The ascent of (Spm + Spd)/Put ratios and descent of Put/PAs ratios showed a positive relation to their salt tolerance. The PAO activity in roots and leaves was all increased, and most obvious in N23232. The relationship between PAs levels in BB52 and its salt tolerance was also discussed.

Oxidoreductases Acting on CH-NH Group Donors↗

Identification and evaluation of O-alkyl substituted hydroxamic acids as potent in vitro inhibitors of the hepatic glycine cleavage system and investigation of their action on in vivo central nervous system glycine concentration.

The identification and evaluation of an extensive series of O-alkyl substituted hydroxamic acids as potent in vitro inhibitors of the hepatic glycine cleavage system is described. An investigation of the action of selected examples on the in vitro brain glycine cleavage system and their influence on in vivo plasma and central nervous system glycine concentrations following systemic administration is also reported.

Animals↗

Solid-state NMR studies of regulation of N-(phosphonomethyl)glycine and glycine metabolism in Pseudomonas sp. strain PG2982.

Lyophilized samples of Pseudomonas sp. PG2982 grown on 13C- and 15N-labeled glyphosate have been analyzed by single and double cross-polarization 13C NMR. Both the carbon and nitrogen metabolism of glyphosate are significantly influenced by the nitrogen source used for the growth of the organism. When ammonium sulfate is the source of nitrogen, the glycyl moiety of glyphosate is utilized intact for the biosynthesis of purines and proteins. But when the organism is grown on glycine as the source of nitrogen, the carbons and nitrogen of glyphosate are scrambled, consistent with incorporation into serine and pyruvate, and hence participation in general metabolism. When both ammonium and glycine are present in the growth medium, regulation of the metabolic fluxes along each of the two major pathways appears to be determined by the intracellular glycine concentration.

Ammonia↗

Benzoyl-coenzyme A:glycine N-acyltransferase and phenylacetyl-coenzyme A:glycine N-acyltransferase from bovine liver mitochondria. Purification and characterization.

Two closely related acyl-CoA:amino acid N-acyl-transferases were purified to near-homogeneity from preparations of bovine liver mitochondria. Each enzyme consisted of a single polypeptide chain with a molecular weight near 33,000. One transferase was specific for benzoyl-CoA, salicyl-CoA, and certain short straight and branched chain fatty acyl-CoA esters as substrates while the other enzyme specifically used either phenylacetyl-CoA or indoleacetyl-CoA. Acyl-CoA substrates for one transferase inhibited the other. Glycine was the preferred acyl acceptor for both enzymes but either L-asparagine or L-glutamine also served. Peptide products for each transferase were identified by mass spectrometry. Enzymatic cleavage of acyl-CoA was stoichiometric with release of thiol and formation of peptide product. Apparent Km values were low for the preferred acyl-CoA substrates relative to the amino acid acceptors (10(-5) M range compared to greater than 10(-3) M). Both enzymes were inhibited by high nonphysiological concentrations of certain divalent cations (Mg2+, Ni2+, and Zn2+). In contrast to benzoyltransferase, phenylacetyltransferase was sensitive to inhibition by either 10(-4) M 5,5'-dithiobis(2-nitrobenzoate) or 10(-5) M p-chloromercuribenzoate; 10(-4) M phenylacetyl-CoA partially protected phenylacetyltransferase against 5,5'-dithiobis(2-nitrobenzoate) inactivation but 10(-1) M glycine did not. For activity, phenylacetyltransferase required addition of certain monovalent cations (K+, Rb+, Na+, Li+, Cs+, or (NH4)+) to the assay system but benzoyltransferase did not. Preliminary kinetic studies of both transferases were consistent with a sequential reaction mechanism in which the acyl-CoA substrate adds to the enzyme first, glycine adds before CoA leaves, and the peptide product dissociates last.

Acyl Coenzyme A↗

Antiplatelet and antithrombotic effects of platelet glycoprotein IIb/IIIa (GPIIb/IIIa) inhibition by arginine-glycine-aspartic acid-serine (RGDS) and arginine-glycine-aspartic acid (RGD) (O-me)Y (SC-46749).

Arginine-glycine-aspartic acid (RGD) is the minimal sequence in fibrinogen that leads to recognition and binding to the glycoprotein IIb/IIIa platelet receptor during aggregation. Analogs of tetrapeptides containing the RGD sequence have been previously shown to block fibrinogen binding to activated platelets in vitro. SC-46749 is an analog of arginine-glycine-aspartic acid-phenylalanine in which the phenylalanine is replaced by O-methyltyrosine. In this study the biological activities of SC-46749 were examined and its actions compared with the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS), one of the natural sequences on the fibrinogen alpha chain that binds to platelets. In vitro, SC-46749 was more potent than RGDS in inhibiting fibrinogen binding (IC50: SC-46749, 27 microM; RGDS, 47 microM), in preventing ADP-induced aggregation in human platelet-rich plasma (IC50: SC-46749, 32 microM; RGDS, 95 microM) and in inhibiting thrombin-induced aggregation in washed human platelets (IC50: SC-46749, 23 microM; RGDS, 64 microM). In rats, SC-46749 prevented collagen-induced thrombocytopenia with an ED50 of 0.87 mg/kg whereas RGDS did not inhibit the response by 50% at doses up to 10 mg/kg. SC-46749 inhibited thrombus formation in an electrically damaged rat carotid artery in a dose-dependent fashion whereas the effects of RGDS were biphasic. RGDS appeared to delay thrombus formation at lower doses but had no effect at higher doses. When infused in dogs for 15 min, SC-46749 prevented ex vivo collagen-induced aggregation at 4 mg/kg/min. These data demonstrate that SC-46749 is a potent inhibitor of platelet aggregation and platelet-dependent thrombus formation.

Animals↗

Stereospecific analysis of triglycerides of Glycine max, Glycine soya, Avena sativa and Avena sterilis strains.

A rapid method for the stereospecific analysis of triglycerides based on enzymatic hydrolysis on thin layer plates was applied to a number of Glycine max, Glycine soya, Avena sativa and Avena sterilis strains. The percentage of each fatty acid on the sn-1-, sn-2- and sn-3-positions was linearly related to the total percentage of the fatty acid in the triglyceride. Large deviations from the common triglyceride pattern were not found.

Dietary Fats↗

Characterization of storage proteins in wild (Glycine soja) and cultivated (Glycine max) soybean seeds using proteomic analysis.

A combined proteomic approach was applied for the separation, identification, and comparison of two major storage proteins, beta-conglycinin and glycinin, in wild (Glycine soja) and cultivated (Glycine max) soybean seeds. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with three different immobilized pH gradient (IPG) strips was an effective method to separate a large number of abundant and less-abundant storage proteins. Most of the subunits of beta-conglycinin were well-separated in the pH range 3.0-10.0, while acidic and basic glycinin polypeptides were well-separated in pH ranges 4.0-7.0 and 6.0-11.0, respectively. Although the overall distribution pattern of the protein spots was similar in both genotypes using pH 3.0-10.0, variations in number and intensity of protein spots were better resolved using a combination of pH 4.0-7.0 and pH 6.0-11.0. The total number of storage protein spots detected in wild and cultivated genotypes was approximately 44 and 34, respectively. This is the first study reporting the comparison of protein profiles of wild and cultivated genotypes of soybean seeds using proteomic tools.

Antigens, Plant↗

Differential effects of ethanol on glycine uptake mediated by the recombinant GLYT1 and GLYT2 glycine transporters.

The effects of ethanol on the function of recombinant glycine transporter 1 (GLYT1) and glycine transporter 2 (GLYT2) have been investigated. GLYT1b and GLYT2a isoforms stably expressed in human embryonic kidney 293 (HEK 293) cells showed a differential behaviour in the presence of ethanol; only the GLYT2a isoform was acutely inhibited. The 'cut-off' (alcohols with four carbons) displayed by the n-alkanols on GLYT2a indicates that a specific binding site for ethanol exists on GLYT2a or on a GLYT2a-interacting protein. The non-competitive inhibition of GLYT2a indicates an allosteric modulation by ethanol of GLYT2a activity. Chronic treatment with ethanol caused differential adaptive responses on the activity and the membrane expression levels of these transporters. The neuronal GLYT2a isoform decreased in activity and surface expression and the mainly glial GLYT1b isoform slightly increased in function and surface density. These changes may be involved in some of the modifications of glycinergic or glutamatergic neurotransmitter systems produced by ethanol intoxication.

Alcohols↗

Timecourse microarray analyses reveal global changes in gene expression of susceptible Glycine max (soybean) roots during infection by Heterodera glycines (soybean cyst nematode).

Changes in gene expression within roots of Glycine max (soybean), cv. Kent, susceptible to infection by Heterodera glycines (the soybean cyst nematode [SCN]), at 6, 12, and 24 h, and 2, 4, 6, and 8 days post-inoculation were monitored using microarrays containing more than 6,000 cDNA inserts. Replicate, independent biological samples were examined at each time point. Gene expression was analyzed statistically using T-tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). These analyses allow the user to query the data in several ways without importing the data into third-party software. RT-PCR confirmed that WRKY6 transcription factor, trehalose phosphate synthase, EIF4a, Skp1, and CLB1 were differentially induced across most time-points. Other genes induced across most timepoints included lipoxygenase, calmodulin, phospholipase C, metallothionein-like protein, and chalcone reductase. RT-PCR demonstrated enhanced expression during the first 12 h of infection for Kunitz trypsin inhibitor and sucrose synthase. The stress-related gene, SAM-22, phospholipase D and 12-oxophytodienoate reductase were also induced at the early time-points. At 6 and 8 dpi there was an abundance of transcripts expressed that encoded genes involved in transcription and protein synthesis. Some of those genes included ribosomal proteins, and initiation and elongation factors. Several genes involved in carbon metabolism and transport were also more abundant. Those genes included glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase and sucrose synthase. These results identified specific changes in gene transcript levels triggered by infection of susceptible soybean roots by SCN.

Animals↗

Laser capture microdissection (LCM) and expression analyses of Glycine max (soybean) syncytium containing root regions formed by the plant pathogen Heterodera glycines (soybean cyst nematode).

Roots of soybean, Glycine max cv. Kent L. Merr., plants susceptible to the soybean cyst nematode (SCN), Heterodera glycines Ichinohe, were inoculated and allowed to develop feeding sites (syncytia) for 8 days. Root samples enriched in syncytial cells were collected using laser capture microdissection (LCM). RNA was extracted and used to make a cDNA library and expressed sequence tags (ESTs) were produced and used for a Gene Ontology (GO) analysis. RT-PCR results indicated enhanced expression of an aquaporin (GmPIP2,2), alpha-tubulin (GmTubA1), beta-tubulin (GmTubB4) and several other genes in syncytium-enriched samples as compared to samples extracted from whole roots. While RT-PCR data showed increased transcript levels of GmPIP2,2 from LCM tissue enriched in syncytial cells, in situ hybridization showed prominent GmPIP2,2 hybridization to RNA in the parenchymal cells tightly juxtaposed to the syncytium. Immunolocalization indicated stronger alpha-tubulin signal within the syncytium as compared to surrounding tissue. However, alpha-tubulin labeling appeared diffuse or clumped. Thus, LCM allowed for the isolation of tissue enriched for syncytial cells, providing material suitable for a variety of molecular analyses.

Amino Acid Sequence↗

Improved synthesis of proline-derived Ni(II) complexes of glycine: versatile chiral equivalents of nucleophilic glycine for general asymmetric synthesis of alpha-amino acids.

A synthetically practical and operationally convenient method for preparing (S)-2-[N-(N'-benzylprolyl)amino]benzophenone (BPBP) and hitherto unknown (S)-2-[N-(N'-benzylprolyl)amino]-4-methylbenzophenone (4-Me-BPBP), (S)-2-[N-(N'-benzylprolyl)amino]-5-nitrobenzophenone (5-NO(2)-BPBP), and their corresponding Ni(II) complexes with glycine [GlyNi(II)BPBP], a widely used chiral equivalent of nucleophilic glycine, and new analogues [GlyNi(II)-4-Me-BPBP] and [GlyNi(II)-5- NO(2)-BPBP] is described. The key step of the method is the synthetically efficient amid bond formation between the corresponding o-aminobenzophenones, featuring significant steric shielding and low nucleophilicity of the amino functionality as well as sterically constrained (S)-N-benzylproline (BP).

Amino Acids↗

Role of glycine-534 and glycine-1179 of human multidrug resistance protein (MDR1) in drug-mediated control of ATP hydrolysis.

The human multidrug resistance protein (MDR1) (P-glycoprotein), a member of the ATP-binding cassette (ABC) family, causes multidrug resistance by an active transport mechanism, which keeps the intracellular level of hydrophobic compounds below a cell-killing threshold. Human MDR1 variants with mutations affecting a conserved glycine residue within the ABC signature of either or both ABC units (G534D, G534V, G1179D and G534D/G1179D) were expressed and characterized in Spodoptera frugiperda (Sf9) cell membranes. These mutations caused a loss of measurable ATPase activity but still allowed ATP binding and the formation of a transition-state intermediate (nucleotide trapping). In contrast with the wild-type protein, in which substrate drugs accelerate nucleotide trapping, in the ABC signature mutants nucleotide trapping was inhibited by MDR1-substrate drugs, suggesting a miscommunication between the drug-binding site(s) and the catalytic domains. Equivalent mutations of the two catalytic sites resulted in a similar effect, indicating the functional equivalence of the two sites. On the basis of these results and recent structural information on an ABC-ABC dimer [Hopfner, Karcher, Shin, Craig, Arthur, Carney and Tainer (2000) Cell 101, 789-800], we propose a key role of these glycine residues in the interdomain communication regulating drug-induced ATP hydrolysis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗