Genetic polymorphisms of Glycine N-acyltransferase (GLYAT) in a French Caucasian population

1. In humans, the glycine N-acyltransferase enzyme (GLYAT) is thought to be important in the detoxification of endogenous and xenobiotic compounds which contain a carboxylic acid group, such as benzoic, isovaleric, or acetylsalicylic acids.

2. The aim of this work was to report a comprehensive investigation of GLYAT genetic polymorphisms in DNA samples from 55 subjects of French Caucasian origin, using polymerase chain reaction–single-strand conformation polymorphism and sequencing strategies.

3. Seven different polymorphisms of the GLYAT gene were identified, including two polymorphisms in the 5′ flanking region of the gene (g.?8457C>T and g.?8010A>G), two polymorphisms in intron 5 (g.13931A>G and g.13944C>T) and three missense mutations in exon 2 (g.49T>A; p.Ser17Thr), exon 5 (g.13886A>G; p.Asn156Ser) and exon 6 (g.14435C>T; p.Arg199Cys). In addition to the wild-type allele GLYAT*1 (2.7%), four novel alleles were identified: GLYAT*2A (75.5%), *2B (4.5%), *3 (16.4%) and *4 (0.9%), and five different genotypes.

4. Localisation of the p.Ser17Thr and p.Arg199Cys missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans.

     

Prolonged zymosan-induced inflammatory pain hypersensitivity in mice lacking glycine receptor alpha2

Glycinergic synapses play a major role in shaping the activity of spinal cord neurons under normal conditions and during persistent pain. However, the role of different glycine receptor (GlyR) subtypes in pain processing has only begun to be unraveled. Here, we analysed whether the GlyR alpha2 subunit might be involved in the processing of acute or persistent pain. Real-time RT-PCR and in situ hybridization analyses revealed that GlyR alpha2 mRNA is enriched in the dorsal horn of the mouse spinal cord. Mice lacking GlyR alpha2 (Glra2^−/− mice) demonstrated a normal nociceptive behavior in models of acute pain and after peripheral nerve injury. However, mechanical hyperalgesia induced by peripheral injection of zymosan was significantly prolonged in Glra2^−/− mice as compared to wild-type littermates. We conclude that spinal GlyRs containing the alpha2 subunit exert a previously unrecognized role in the resolution of inflammatory pain.     

Paradoxical increase in seizure frequency with valproate in nonketotic hyperglycinemia

Nonketotic hyperglycinemia (NKH), or glycine encephalopathy, is an autosomal recessive disorder caused by a defect in the glycine cleavage enzyme system. In neonatal-onset NKH, patients manifest lethargy, hypotonia, apnea, and intractable epileptic seizures that are not specific to this disease. We experienced a 6-year-old girl with spastic quadriplegia, intractable epilepsy, and mental retardation, all initially regarded as sequelae of neonatal meningitis. The seizure frequency was transiently increased when valproate was started. Head MRI revealed progressive brain atrophy and white matter loss with high intensity signals on T2-weighted and diffusion-weighted images, which prompted us to conduct further metabolic workups. High glycine levels led us to suspect NKH, and we confirmed this diagnosis by the non-invasive, ¹³C-glycine breath test. DNA sequencing revealed novel Leu885Pro/Trp897Cys mutations in the glycine decarboxylase gene that were transmitted from both parents. Sodium benzoate and dextromethorphan dramatically decreased her hypertonicity. Our case shows that paradoxical increases in seizure frequency following valproate can be a clue for a diagnosis of NKH, and that a correct diagnosis of NKH can greatly alter the quality of life in such patients.     

A lethal and rare cause of arthrogryposis: Glyt1 encephalopathy

Glycine encephalopathy with normal serum glycine (MIM #617301), also known as GLYT1 encephalopathy, is an extremely rare disorder caused by biallelic variants in SLC6A9 and characterised by facial dysmorphic features, skeletal findings including contractures, knee hyperextension, and joint dislocations and seizures. To date, only ten patients from five families have been reported and only two of them could survive until childhood. In this study, we report on a consanguineous Turkish couple with a history of six pregnancies with three habitual abortions and three postpartum exitus. While in three pregnancies the babies were born prematurely at 32nd gestational week by emergency ceserean section due to hydrops and fetal distress, the other pregnancy was medically terminated at 16th gestational week due to absent fetal heart activity. The product of all these three pregnancies exhibited similar phenotype including short neck, thoracic kyphosis, hypertrichosis, joint contractures and dislocations, hypertonia, knee hyperextension and facial dysmorphic features. Trio exome sequencing was performed prenatally during the last pregnancy and a novel VUS variant in SLC6A9 and a likely pathogenic variant in MTOR gene were detected. DNA isolation was performed from frozen muscle and adrenal tissue of previously autopsied fetuses with similar clinical features, and the same variants were confirmed in both of them. Our data suggest that SLC6A9 and MTOR variants may be responsible for this extremely lethal phenotype in this family.     

Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen

Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.     

Glycine induces a novel form of long-term potentiation in the superficial layers of the superior colliculus

1. The mammalian superior colliculus (SC) is a midbrain nucleus containing space maps of different sensory modalities which show various forms of age- and activity-dependent plasticity in vivo and in vitro. In the present study, we aimed to characterize the role of glycine (Gly) receptors in the SC, and we observed that application of glycine (Gly; 500 μM and 3 mM) for 7 min to SC slices of adult guinea-pigs caused a novel form of long-term potentiation (termed LTP_gly) of evoked excitatory postsynaptic potentials recorded in the superficial layers.
2. The strength of potentiation was found to be concentration-dependent and partially independent from synaptic stimulation.
3. LTP_gly did not involve NMDA receptor activation as proven by the lack of inhibition by 100 μM D,L-2-amino-5-phosphonovaleric acid (APV) and 10 μM MK-801.
4. LTP_gly could only be masked but not prevented by strychnine (100 μM) and remained undisturbed in the presence of picrotoxin (100 μM).
5. Inhibition of carbonic anhydrase by acetazolamide (20 μM) had no effect on LTP_gly suggesting that the excitatory action of Gly is not due to a differential breakdown of the Cl⁻/HCO₃⁻ gradients.
6. As indicated by the inhibition of LTP_gly of the fEPSP slope by the L-type calcium channel blocker nifedipine (20 μM), voltage-dependent calcium channels are the source for Ca²⁺ elevation as the intracellular trigger.
7. Our data provide the first evidence for a role of Gly in SC synaptic transmission. They illustrate a so far unknown action of Gly which can lead to long-lasting changes of synaptic efficacy and which is not mediated via NMDA-related or strychnine-sensitive binding sites.

     

Actions of amino acids and convulsants on bulbar reticular neurones

Summary A study has been made of the actions of microelectrophoretically administered amino acids and convulsants on spontaneous and glutamate- (or DLH-) induced firing of bulbar reticular neurones in unanaesthetized, decerebrate, cats. DLH was a more potent excitant than glutamate and aspartate on almost all the neurones tested. Although glutamate was usually more effective than aspartate, their relative potencies were often similar. Glycine, -alanine and GABA depressed the majority of neurones tested (93%, 89% and 75%, respectively), and had no action on the remainder. Glycine was invariably more potent than -alanine, which was usually more potent than GABA. Strychnine reversibly blocked the depressant actions of glycine and -alanine but not that of GABA. Electrophoretically administered picrotoxin slightly reduced the depression caused by glycine and GABA on less than half the neurones tested. Intravenously injected picrotoxin (0.3–5 mg/kg) did not block the effects of these amino acids. A comparison of the results with those obtained in the spinal cord provides some evidence that glycine may be an inhibitory transmitter substance released on bulbar reticular neurones.Supported by a grant from the Swiss National Foundation of Scientific Research.     

The inhibition of hypoglossal motoneurones by impulses in the glossopharyngeal nerve of the rat

Summary Stimulation of the glossopharyngeal nerve produced inhibitory post synaptic potentials in neurones of the hypoglossal nucleus of the rat. The corresponding pause in the amino acid induced firing of these cells was antagonized by electrophoretically administered strychnine but not by bicuculline. Strychnine and bicuculline antagonized the actions of glycine and GABA respectively on these cells. The findings suggest that the transmitter for this synaptic inhibition is a glycine-like amino acid.C. J. Martin Travelling Fellow.Wellcome Trust/Animal Health Trust Research Fellow.     

Glycine-like immunoreactivity in the cerebellum of rat and Senegalese baboon,Papio papio: a comparison with the distribution of GABA-like immunoreactivity and with [3H]glycine and [3H]GABA uptake

Summary An antiserum against conjugated glycine was characterized and applied to cerebellar sections of rats and baboons that had been perfusion-fixed with glutaraldehyde. After immunosorbent purification the serum reacted with brain protein-glutaraldehyde-glycine conjugates, but did not stain similar test conjugates prepared from other amino acids, including GABA and -alanine. In the rat cerebellum the glycine antiserum selectively labelled a subpopulation of Golgi neurons. Adjacent Vibratome sections treated with an antiserum against conjugated GABA revealed an about equally large subpopulation of immunopositive Golgi cells. A proportion of the Golgi cells that were cleaved by the plane of section contained both immunoreactivities. Additional evidence for a colocalization of glycine and GABA was obtained by postembedding staining of alternate semithin sections with the GABA antiserum and glycine antiserum, respectively. The ability of the antisera to distinguish between fixed glycine and GABA was corroborated by preincubation of the antisera with glutaraldehyde-amino acid fixation complexes: glycine complexes abolished staining with the glycine antiserum but had no effect on the GABA antiserum. The opposite effects were obtained with the GABA complexes. Matching the distributions of the respective immunoreactivities, [³H]glycine uptake was restricted to glomerulus-like structures in the granule cell layer whereas [³H]GABA uptake also occurred in punctate and fibrous profiles in the molecular layer. The baboon showed a distribution of glycine-like immunoreactivity similar to that in the rat, except that a few immunopositive neurons occurred in the molecular layer. The latter neurons were interpreted as outlying Golgi neurons; however, the possibility that they represent a subpopulation of basket cells could not be excluded. The Purkinje cells were negative in both species. Glial cells were weakly stained with the glycine antiserum but were strongly immunopositive after incubation with an antiserum raised against conjugates of the structurally similar amino acid -alanine. The present data suggest that glycine and GABA occur in about equally large subpopulations of Golgi neurons. A subpopulation of the Golgi neurons appears to contain both glycine and GABA.     

A pharmacological study of the depression of spinal neurones by glycine and related amino acids

Summary An analysis has been made in anaesthetised cats of the depression by glycine and related amino acids of the firing of spinal dorsal horn interneurones, Renshaw cells and cortical neurones. In general, electrophoretically administered glycine was a more potent depressant of interneurones than GABA. The reverse was true for cortical neurones, whereas these two amino acids were approximately equally effective upon Renshaw cells. Strychnine blocked the depressant action of - and -amino acids, but not that of - and higher -amino acids. Only convulsants having a strychnine-like effect on spinal post-synaptic inhibition blocked the action of glycine. The depression of spinal neurones produced by glycine or GABA was not affected by structural analogues of glycine and GABA that were not depressants, or by substances influencing amino acid transport systems. Some evidence was obtained for the enzymic inactivation of electrophoretically administered glycine in spinal tissue. The results are discussed in terms of the involvement of a glycine-like amino acid as a major spinal inhibitory transmitter.Supported by a grant from the Swiss Academy of Medical Sciences.