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Effect of glutamic acid diethylester on (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainic acid-induced changes of body temperature in rats.

A low dose (1 microgram) of intracerebroventricularly injected (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) produced a hyperthermic response in rats, while a high dose of AMPA (2.5 micrograms), similarly to kainic acid (0.1 microgram) produced a biphasic effect: short-lasting hypothermia followed by hyperthermia. These effects on body temperature were not affected by pretreatment with 200 mg/kg ip of glutamic acid diethylester (GDEE), which by itself, produced a significant hypothermia. The results indicate that the effects of kainic acid and AMPA on body temperature are not mediated by GDEE-sensitive glutamate receptors.

Animals↗

Induction of T lymphocyte responses to a small molecular weight antigen. I. Failure to induce tolerance in azobenzenearsonate (ABA)-specific T cells in guinea pigs with an ABA conjugate of A copolymer of D-glutamic acid and D-lysine (D-GL).

2,4-Dinitrophenyl (DNP) coupled to the copolymer D-glutamic acid and D-lysine (D-GL) induces B cell tolerance but not T cell tolerance. This implies either a lack of DNP determinant recognition by T cells or a substantial difference in tolerance mechanisms for the two cell types. In the present study D-GL was conjugated with the well-defined determinant azobenzenearsonate (ABA) coupled to single amino acids shown here and previously by others to trigger effectively T lymphocytes. The experiments presented here demonstrate that these ABA conjugates of D-GL, although capable of diminishing anti-ABA antibody production, completely fail to render ABA-specific T lymphocytes tolerant thus drawing us to conclude that there are significant operational differences in the mechanisms of tolerance induction in T and B lymphocytes, respectively.

Animals↗

A pore-lining glutamic acid in the rat olfactory cyclic nucleotide-gated channel controls external spermine block.

Spermine is a potent, voltage-dependent blocker of the olfactory cyclic nucleotide-gated channel from both the intracellular and extracellular sides. However, its sites of action are unknown. This study investigated the external spermine binding site in the rat CNCalpha3 subunit. Neutralization of a glutamic acid residue (E342Q) in the P-loop region eliminated voltage-dependence of block by externally applied spermine. The charge-conservative E342D mutation had little effect on spermine block. Thus, E342 forms the binding site for externally applied spermine. However, spermine remained a potent voltage-independent blocker of the E342Q mutant channel, suggesting that the mutation either created a novel binding site outside the membrane electrical field or that it dramatically changed the properties of the existing pore site.

Animals↗

Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose.

It has been shown previously that externally added glycine betaine is accumulated in Escherichia coli in response to the external osmotic strength. Here we have shown, by using nuclear magnetic resonance spectroscopy and radiochemical methods, that E. coli growing in a glucose-mineral medium of elevated osmotic strength generated with NaCl, had the same capacity to accumulate proline betaine and glycine betaine. Its capacity to accumulate gamma-butyrobetaine was, however, 40 to 50% lower. Accordingly, externally added proline betaine and glycine betaine stimulated aerobic growth of osmotically stressed cells equally well, and they were more osmoprotective than gamma-butyrobetaine. In cells grown at an osmotic strength of 0.64, 1.01, or 1.47 osmolal, respectively, the molal cytoplasmic concentration of the two former betaines corresponded to 29, 38, or 58% of the external osmotic strength. Nuclear magnetic resonance spectroscopy revealed that trehalose and glutamic acid were the only species of organic osmolytes accumulated in significant amounts in cells grown under osmotic stress in glucose-mineral medium without betaines. Their combined molal concentration in the cytoplasm of cells grown at 1.01 osmolal corresponded to 27% of the external osmotic strength.

Amino Acids↗

Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).

Amino Acid Sequence↗

Oxytocin and vasopressin release in the olfactory bulb of parturient ewes: changes with maternal experience and effects on acetylcholine, gamma-aminobutyric acid, glutamate and noradrenaline release.

Maternal behaviour and the ewe's ability to recognize her lamb depend on olfactory cues and parturition, and are facilitated by maternal experience. Parturition induces a variety of neurochemical changes in the brain and, in particular, oxytocin (OT) release. This peptide injected centrally induces maternal behaviour. Oxytocin release occurs in the olfactory bulb (OB) at parturition and yet this structure is involved in the process of selective bonding with lamb. The present study therefore investigated the possibility that oxytocin release in the OB might modulate the release of classical transmitters that are known to be important in controlling selective recognition and whether maternal experience has any effect on this. We have first used in vivo microdialysis to measure OT release, as well as that of the related peptide, arginine-vasopressin (AVP), in the OB of maternally experienced and inexperienced ewes during parturition. While OT release significantly increased in both primiparous and multiparous ewes at parturition this increase was significantly greater in multiparous ewes. No significant change of AVP release was observed in either group. However, vagino-cervical stimulation (VCS) performed at 6 h post-partum caused similar increases in OT but not AVP release in both primiparous and multiparous ewes suggesting that the first birth experience potentiates the ability of VCS to evoke OT release within 6 h of parturition. Using retrodialysis, either OT (10 microM) or AVP (10 microM) were infused into the OB of multiparous and nulliparous ewes and their effects on modulating acetylcholine (ACh), noradrenaline (NA), glutamate and gamma-aminobutyric acid (GABA) release were monitored. Both peptides produced an increase of ACh and NA in multiparous animals and this effect was either absent or less pronounced in nulliparous animals. OT, but not AVP, also increased GABA release equivalently in nulliparous and multiparous animals. Glutamate release was not altered in response to OT or AVP infusion. These results suggest that OT release in the OB at parturition may facilitate the recognition of lamb odours by modulating NA, ACh and GABA release which are of primary importance for olfactory memory. The reduced release of OT in the OB of primiparous ewes at parturition, together with its reduced ability to modulate NA and ACh release, might also partly explain why maternally inexperienced animals require a longer period to selectively bond with their lambs.

Acetylcholine↗

Glutamic acid decarboxylase autoantibodies in controlled and uncontrolled epilepsy: a pilot study.

There have been anecdotal reports of raised glutamic acid decarboxylase (GAD) autoantibodies in patients with refractory epilepsy. We measured serum GAD autoantibodies in 105 patients with idiopathic or symptomatic epilepsy. There was no significant difference in the absolute titre of GAD autoantibody between patients with controlled and uncontrolled epilepsy. However, four female patients with uncontrolled epilepsy had levels that were over three times above the highest detected in the seizure-free group, three of whom also tested positive for pancreatic islet cell antibodies. Larger scale studies, perhaps comparing different epilepsy syndromes, are required to determine the exact clinical role of GAD autoantibodies in epilepsy.

Adolescent↗

Potentiation by glutamic acid dimethyl ester of GLP-1 insulinotropic action in fed anaesthetized rats.

The dimethyl ester of L-glutamic acid (DMG) stimulates insulin release and was proposed as a possible insulinotropic tool in the treatment of non-insulin-dependent diabetes. In such a perspective, it was investigated whether DMG enhances the B-cell secretory response to GLP-1 in fed anaesthetized rats. The primed constant infusion of DMG (1.0 micromol and then 0.5 micromol/min, both per g body wt.) provoked a rapid and sustained increase in plasma insulin concentration and augmented the release of insulin caused by GLP-1. Thus, DMG indeed appears as a suitable tool to potentiate the insulinotropic action of GLP-1.

Anesthesia↗

Elevated immunoreactivity for glutamic acid decarboxylase in the rat cerebral cortex following transient middle cerebral artery occlusion.

We investigated the expression of glutamic acid decarboxylase (molecular weight 67,000; GAD67) immunohistochemically in the rat cerebral cortex following transient middle cerebral artery occlusion (MCAO) capable of producing slowly progressive neuronal damage. An increase in GAD67 immunoreactivity was observed in the cerebral cortex ipsilateral to the ischemic insult, most prominent in lamina IV, 3 to 14 days after MCAO. At this stage, light microscopy showed GAD67-positive puncta to be larger and more strongly immunoreactive in the ipsilateral cortex than those in the contralateral side. The elevated expression of GAD67 in the insulted cortex may reflect part of the adaptive functional changes in GABA transmission with slowly progressive cortical ischemic damage.

Animals↗

Alterations in expression of messenger RNAs encoding two isoforms of glutamic acid decarboxylase in the globus pallidus and entopeduncular nucleus in animals symptomatic for and recovered from experimental Parkinsonism.

Glutamic acid decarboxylase (GAD65, GAD67) mRNA expression was measured in the globus pallidus (GP) and entopeduncular nucleus (ENTO) of normal, and MPTP-lesioned cats symptomatic for and recovered from MPTP-induced Parkinsonism. In the ENTO of symptomatic cats, GAD65 and GAD67 mRNA expression were both significantly increased, while only GAD67 gene expression was increased in the GP. Levels of gene expression for both isoforms were normal in the GP and ENTO of spontaneously recovered animals. Increased expression of GAD65/67 mRNA in the ENTO corresponded with expression of Parkinsonian signs, suggesting a contribution of both isoforms to ENTO functioning and perhaps a greater contribution of GAD67 expression to GP functioning.

Animals↗

Glutamic acid decarboxylase in sea lamprey (Petromyzon marinus): characterization, localization, and developmental changes.

We have carried out assays for glutamic acid decarboxylase (GAD) in homogenates of brain and spinal cord from larval and adult sea lamprey (Petromyzon marinus). The enzyme had similar characteristics in both stages. Optimal pH was 6.8; optimal temperature was 27-30 degrees C; Km at 27 degrees C was 5 mM. GAD activity was distributed uniformly along the length of the spinal cord. Specific activities for the larval cord and brain were 26 and 63 nm CO2/mg protein/h, respectively. The specific activities for the adult cord and brain were 29 and 236 nm CO2/mg protein/h, respectively. Thus, the activity of cord homogenates did not change significantly between larval and adult stages, but that of the brain increased about fourfold.

Aging↗

Downbeating nystagmus and muscle spasms in a patient with glutamic-acid decarboxylase antibodies.

PURPOSE: To report the ophthalmic findings and response to treatment in a patient with glutamic-acid decarboxylase antibodies. DESIGN: Case report. METHODS: A 55-year-old woman developed progressive, painful, low back muscle spasms, vertical diplopia, downbeating nystagmus, and asymmetric appendicular ataxia. RESULTS: Downbeating nystagmus was present in primary gaze with an alternating skew deviation in lateral gaze. Serum and cerebrospinal fluid GAD antibodies were detected. Treatment with diazepam led to resolution of spasticity, whereas repeated courses of intravenous immunoglobulin improved cerebellar function, including appendicular ataxia and downbeating nystagmus. CONCLUSIONS: Patients with GAD antibodies may have elements of both Stiff-person syndrome (muscle rigidity and spasms) and prominent cerebellar dysfunction. Treatment with diazepam rapidly improved Stiff-person symptoms, whereas IVIg was partially effective at the early stage of cerebellar dysfunction.

Autoantibodies↗

[Antibodies to glutamic acid decarboxylase in patients with Graves disease].

OBJECTIVE: To investigate the level and clinical implication of antibodies to glutamic acid in patients with Graves disease. METHODS: ELISA and RIA were used to determine the decarboxylase (GADAb) levels of GADAb, thyroglobulin antibodies (TGAb), thyroidperoxidase antibodies(TPOAb), free triiodothyronine (FT3) and free thyroxine (FT4) before and after treatment in the patients with Graves disease not yet complicated by diabetes mellitus and in the healthy volunteers as controls. Then the relations of GADAb to FT3, FT4, TGAb and TPOAb were detected. RESULTS: The correlation coefficients between GADAb and FT3, FT4 were--0.367 (P > 0.05) and 0.029 (P > 0.05), respectively. The correlation coefficients between GADAb and titers of TGAb, TPOAb were 0.320 (P > 0.05) and 0.394 (P > 0.05), respectively. The levels of GADAb, TGAb, TPOAb, and the ratio of patients with positive GADAb were reduced in the treated patients with Graves disease, but no significant difference was observed between the treated and untreated patients. CONCLUSION: The above data indicated that GADAb was related to Graves disease, but no relation was seen between GADAb and thyroid

Adult↗

Bicyclic glutamic acid derivatives.

For the second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids via Strecker reaction of chiral ketimines, the cyanide addition as the key stereodifferentiating step produces mixtures of diastereomeric alpha-amino nitrile esters the composition of which is independent of the reaction temperature and the type of the solvent, respectively. The subsequent hydrolysis is exclusively achieved with concentrated H(2)SO(4) yielding diastereomeric mixtures of three secondary alpha-amino alpha-carbamoyl-gamma-esters and two diastereomeric cis-fused angular alpha-carbamoyl gamma-lactams as bicyclic glutamic acid derivatives, gained from in situ stereomer differentiating cyclisation of the secondary cis-alpha-amino alpha-carbamoyl-gamma-esters. Separation was achieved by CC. The pure secondary trans-alpha-amino alpha-carbamoyl-gamma-esters cyclise on heating and treatment with concentrated H(2)SO(4), respectively, to diastereomeric cis-fused angular secondary alpha-amino imides. Their hydrogenolysis led to the enantiomeric cis-fused angular primary alpha-amino imides. The configuration of all compounds was completely established by NMR methods, CD-spectra, and by X-ray analyses of the (alphaR,1R,5R)-1-carbamoyl-2-(1-phenylethyl)-2-azabicyclo[3.3.0]octan-3-one and of the trans-alphaS,1S,2R-2-ethoxycarbonylmethyl-1-(1-phenylethylamino)cyclopentanecarboxamide.

Amino Acids, Cyclic↗

Development of glutamic acid decarboxylase-immunoreactive elements in the cerebellar cortex of normal and lurcher mutant mice.

The development of glutamic acid decarboxylase-immunoreactivity (GAD-IR) in cells, fibers, and varicosities of the cerebellar cortex has been examined by light microscopy in normal and lurcher mutant mice between postnatal day 3 and 30 (P3-P30). Purkinje cell morphology was demonstrated in adjacent sections by using an antiserum to the 28Kd vitamin D-dependent calcium binding protein (CaBP). In early postnatal lurcher mice, but not in normal littermates, GAD-IR fibers, presumably Purkinje cell pseudopodia, invade the external granular layer. The plexus of CaBP-IR axons in the internal granular layer is much less complex in lurcher mice than in normal littermates, even before the onset of lurcher Purkinje cell degeneration at P8. In normal mice, GAD-IR fibers encapsulate Purkinje cell somata by P15. Lurcher Purkinje cells, in contrast, receive scattered contacts by GAD-IR puncta and possess a "cap" of such elements surrounding the primary dendrite and apical soma. Pinceau formations, visible as a knot of GAD-IR puncta hanging from the base of Purkinje cells in normal P15 mice, are not present in lurcher littermates. "Empty baskets" or collapsed pinceau formations in regions devoid of Purkinje cells are not revealed by anti-GAD immunohistochemistry in the P17-P30 lurcher cerebellar cortex.

Animals↗

TRH stimulation and L-glutamic acid inhibition of proximal gastric motor activity in the rat dorsal vagal complex.

The importance of the dorsal vagal complex (DVC) in the control of gastric motor activity has been previously established by electrical and chemical stimulation of this region. We have further evaluated excitatory and inhibitory influences on motor activity of the gastric corpus by microinjection of L-glutamic acid (GLU) and thyrotropin-releasing hormone (TRH) into the DVC. GLU and TRH were ejected by pressure (20-30 psi) in 1-10 nl vol. from multibarreled micropipettes and intraluminal pressure in the gastric corpus was measured using a manometric catheter placed into the stomach through the pylorus of urethane-chloralose anesthetized rats. Gastric motor activity was monitored while micropipettes were advanced from the surface of the dorsomedial medulla to a depth of 1 mm in 100 micron increments. Microinjections of GLU (1-10 pmol) at depths of 200-600 microns below the surface of the brainstem caused a decrease in tonic intraluminal pressure and amplitude of phasic contractions of the gastric corpus. Injection of TRH (1-10 pmol) at depths of 200-800 microns increased both tonic intraluminal pressure and amplitude of phasic contractions. The responses to GLU (10 pmol) and TRH (10 pmol) were abolished by hexamethonium and vagotomy; atropine abolished the effect of TRH and attenuated that of GLU. It is concluded that GLU evokes only vagally mediated inhibitory effects on tonic and phasic gastric motor activity when microinjected into the DVC. In contrast, injection of TRH at the same loci causes only vagal cholinergic increases in motor activity. Subpopulations of neurons in the DVC may, therefore, be activated by specific neurotransmitters having opposite effects on gastric motor activity.

Animals↗

Lack of antibodies to glutamic acid decarboxylase in young adults of the high diabetes prevalence Wanigela people of Papua New Guinea.

Antibodies to glutamic acid decarboxylase (anti-GAD) are common in typical insulin-dependent diabetes mellitus, and also identify a sub-group of older persons who are originally misdiagnosed as having non-insulin-dependent disease (NIDDM). The Wanigela people of Papua New Guinea are highly susceptible to diabetes mellitus, with a prevalence of 20.4% in urbanised young adults aged 25-34 years. On the basis of clinical features including the presence of obesity and relatively high insulin concentrations the Wanigelas have NIDDM. To determine whether anti-GAD is present in this high prevalence form of diabetes, and to investigate whether there might be an autoimmune component to the disease, we measured anti-GAD in 93 newly-diagnosed diabetic subjects aged 25-44 years, and in 40 controls with normal glucose tolerance. There was no difference in mean levels of anti-GAD in diabetic subjects and normal controls. Two subjects had borderline elevated anti-GAD levels: one was a normal control, and the other a diabetic. This study shows that anti-GAD is not present in this (and probably other) high prevalence variant of NIDDM. Moreover, the results suggest strongly that diabetes in the Wanigela people is unlikely to have an autoimmune component to its pathogenesis.

Adult↗