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PubMed1997

Expression of recombinant human glutamic acid decarboxylase (GAD) in myeloma cells and enzyme-linked immunosorbent assay (ELISA) for autoantibodies to GAD.

Detection of serum autoantibodies to glutamic acid decarboxylase (GAD) is a new method to differentiate insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). We established a transformed mouse myeloma cell line, SPG14, which expresses recombinant human GAD65, a major isomer of 65 kDa, inside the cells. GAD65 was partially purified by affinity chromatography using the mouse anti-GAD antibody (Ab). We also established a sandwich ELISA for anti-GAD Ab of the IgG class using GAD65 for coating and the anti-human IgG for detection and examined 54 sera of the IDDM patients and 45 sera of normal individuals. When the mean +2 SD of the color development of the sera of normal individuals was used as a cut-off level, 59.2% of patients with IDDM were positive. This indicates that the ELISA was effective to differentiate IDDM and NIDDM. The result also indicates that the autoantibody to GAD does not dissociate from the recombinant GAD rapidly, even when unbound anti-GAD Ab was fully removed. By using a perfusion culture system, we obtained as many as 4.2 x 10(10) SPG14 cells, from which 5 mg of GAD65 could be obtained; this is sufficient for 5,000 assays. This system could be clinically useful for large-scale diagnosis of IDDM.

Animals↗
PubMed2005

Characterization of the humoral immune response to glutamic acid decarboxylase in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and/or type 1 diabetes.

OBJECTIVE: A humoral autoimmune response to glutamic acid decarboxylase (GAD65) is common both in patients with type 1 diabetes and in those with the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, while overt type 1 diabetes is relatively rarely diagnosed in APECED patients. The aim of this study was to assess whether this difference in the incidence of type 1 diabetes is associated with variability in the humoral immune response to GAD65, one of the major autoantigens in type 1 diabetes. METHODS: Epitope- and isotype-specific GAD65 autoantibodies were analysed in 20 patients with APECED and 20 patients with newly diagnosed type 1 diabetes alone by radiobinding assays. RESULTS: GAD65 autoantibodies targeted the middle and carboxy-terminal regions of GAD65 and occasionally the amino-terminal region in the APECED patients and comprised mainly the IgG1 subclass and less frequently the IgG2 and IgG4 subclasses. The profile of epitope- and isotype-specific GAD65 autoantibodies was similar in type 1 diabetes and APECED, except that IgG2 subclass antibodies were observed more often and at higher levels in the patients with type 1 diabetes alone (P < 0.05). None of the measured parameters separated APECED patients with type 1 diabetes from those without type 1 diabetes. CONCLUSION: APECED-associated humoral autoimmunity to GAD65 does not differ markedly from that observed in type 1 diabetes; only IgG2-GAD65 antibodies may be more closely associated with the latter entity.

Adult↗
PubMed1993

HLA-DQ genotypes are associated with autoimmunity to glutamic acid decarboxylase in insulin-dependent diabetes mellitus patients.

This study has investigated the genetic basis of the heterogeneous autoimmune response to glutamic acid decarboxylase (GAD) in 179 Australian patients with IDDM. Antibodies to GAD have been correlated with HLA-DQB1 alleles and genotypes, as determined by sequence-specific oligonucleotide hybridizations after polymerase chain reaction was applied to exon 2 of the DQ beta 1 gene. HLA-DQ2 was significantly increased (p < 0.01) in IDDM patients with antibodies to GAD. Antibodies to GAD were detected in 64% of 72 DQ2.8 patients, in 55% of 29 DQ2.2 or DQ8.8 patients and in 41% of 78 patients with other HLA-DQB1 genotypes. HLA-DQ genotype association with autoimmunity to GAD was statistically significant (p = 0.02) and reflected early formation of antibodies to GAD, rather than an HLA association with persistence of antibodies to GAD, since the genotype effect was more evident (p = 0.02) in those with more recent onset (0-5 years) of IDDM. Also, the HLA-DQ genotype effect was more evident in patients with IDDM onset after the age of 14 years (p = 0.003). Multivariate analysis showed that HLA-DQB1 genotypes had a more significant impact on antibodies to GAD than either duration or age of onset of IDDM. In patients with IDDM in childhood, only a minority had low-risk HLA-DQB1 genotypes (37%) when compared with those with onset in adulthood (62%) (p = 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2005

Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD(500-585).

Tolerance induction of autoreactive T cells against pancreatic beta cell-specific autoantigens such as glutamic acid decarboxylase 65 (GAD65) and insulin has been attempted as a method to prevent autoimmune diabetes. In this study, we investigate whether adenoassociated virus (AAV) gene delivery of multiple immunodominant epitopes expressing GAD(500-585) could induce potent immune tolerance and persistently suppress autoimmune diabetes in NOD mice. A single muscle injection of 7-wk-old female NOD mice with rAAV/GAD(500-585) (3 x 10(11) IU/mouse) quantitatively reduced pancreatic insulitis and efficiently prevented the development of overt type I diabetes. This prevention was marked by the inactivation of GAD(500-585)-responsive T lymphocytes, the enhanced GAD(500-585)-specific Th2 response (characterized by increased IL-4, IL-10 production, and decreased IFN-gamma production; especially elevated anti-GAD(500-585) IgG1 titer; and relatively unchanged anti-GAD(500-585) IgG2b titer), the increased secretion of TGF-beta, and the production of protective regulatory cells. Our studies also revealed that peptides 509-528, 570-585, and 554-546 in the region of GAD(500-585) played important roles in rAAV/GAD(500-585) immunization-induced immune tolerance. These data indicate that using AAV, a vector with advantage for therapeutic gene delivery, to transfer autoantigen peptide GAD(500-585), can induce immunological tolerance through active suppression of effector T cells and prevent type I diabetes in NOD mice.

Animals↗
PubMed1998

Serotonergic regulation of neuropeptide and glutamic acid decarboxylase mRNA levels in the rat striatum and globus pallidus: studies with fluoxetine and DOI.

The serotonergic regulation of neuropeptide and glutamic acid decarboxylase (GAD) mRNA level in the rat basal ganglia was investigated by determining the effects of chronic treatment with the serotonin uptake blocker fluoxetine and the serotonin 5-HT2 agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrobromide (DOI). Fluoxetine (10 mg/kg) induced a reduction of preproenkephalin and GAD65 mRNA levels in the caudate-putamen and nucleus accumbens core and shell after 5 days of treatment. In addition, GAD65 mRNA levels were reduced in the globus pallidus. These changes appeared to be transient as they were not found after 15 days of fluoxetine treatment. DOI (7 mg/kg), administered for 9 days, induced a decrease of preprodynorphin mRNA levels in the caudate-putamen and the nucleus accumbens core and shell. No regional differentiation in the effects of fluoxetine and DOI was observed. Based on the present results, we propose that an increased 5-HT tone may reduce enkephalin and GABA mRNA levels in striatal regions and in the globus pallidus. Our results further show that preproenkephalin mRNA is not affected by chronic 5-HT2 receptor stimulation, indicating that the fluoxetine-induced decrease in preproenkephalin mRNA levels involves other 5-HT receptors than the 5-HT2 receptor. Preprodynorphin mRNA levels, on the other hand, were found to be reduced after chronic 5-HT2 receptors than stimulation. This observation, together with our previous finding that the 5-HT2 antagonist ritanserin tends to increase preprodynorphin mRNA levels, suggests a 5-HT2-mediated tonic inhibition of preprodynorphin mRNA levels.

Amphetamines↗
PubMed1997

Glutamic acid decarboxylase65 (GAD65) antibodies and insulin auto-antibodies in Japanese patients with non-insulin-dependent diabetes mellitus.

To clarify whether glutamic acid decarboxylase65 antibodies (GAD65 Ab) and insulin autoantibodies (IAA) are good predictive markers for insulin-dependency in NIDDM, we studied GAD65 Ab and IAA in NIDDM patients treated with diet alone or in combination with oral hypoglycemic agents. GAD65 Ab were found in 12 or 29 (5.2%, P = 0.079 vs. control) NIDDM patients and IAA in 8 of 229 (3.5%). The frequency of GAD65 Ab and IAA positivity in NIDDM did not differ significantly from those of healthy controls (2/150, 1.3%, 2/150, 1.3%, respectively), but the frequency of patients who were positive for either GAD65 Ab or IAA, or both, was significantly higher than that of normal controls (17/229, 7.4% and 4/150, 2.7%, respectively, P < 0.05). In addition, the prevalences of GAD65 Ab and of IAA in those patients whose disease durations, since the diagnosis of diabetes, were less than one year were significantly higher than those of controls (4/30, 13.3%, P < 0.05, 4/30, 13.3%, P < 0.05, respectively). We found no differences between GAD65 Ab positive- and negative-patients in either BMI or serum C-peptide levels. Over a one to five year follow-up period (mean 2.0 yrs), serum C-peptide levels gradually decreased necessitating insulin treatment in three of the patients positive for GAD65 Ab and/or IAA (3/17, 17.6%; two were positive for both GAD65 Ab and IAA and one was positive for GAD65 Ab only). In contrast, only five patients negative for the two antibodies developed insulin requirement (5/212, 2.4%, P < 0.01). These results suggest that GAD65 Ab and IAA are good markers for predicting the development of insulin dependency in NIDDM patients and that the predictive value for insulin-dependency in NIDDM is enhanced by measuring both antibodies.

Adolescent↗
PubMed1975

Molecular structure--activity relationship of hydrazides inhibiting glutamic acid decarboxylase, GABA-alpha-oxoglutarate aminotransferase, and monoamine oxidase activities in chick brain.

Several aryl and heteroaryl hydrazides were synthesized and evaluated for their inhibitory effects on glutamic acid decarboxylase (GAD), GABA-alpha-oxoglutarate aminotransferase (GABA-T), and monoamine oxidase (MAO) enzyme systems in chick brain 24 h after their intramuscular administration (0.75 mmol/kg). All compounds produced a reduction in GAD, GABA-T, and MAO activity. Structure-activity relationships indicated that the ring structure had a greater influence on the degree of GAD and GABA-T inhibition than did the N'-terminal group. In contrast, structural requirements for MAO inhibition were much more restrictive. The intramuscular administration of benzoic acid hydrazide to chicks 24 h prior to their being exposed to oxygen at high pressure provided significant protection against the onset of the hyperbaric oxygen-induced seizures.

Animals↗
PubMedDate not supplied

Effect of aging on lipid peroxide levels induced by L-glutamic acid and estimated by means of a thiobarbituric acid test in rat brain tissue.

Studies of age-related changes based on the thiobarbituric acid (TBA) test appear to be inconsistent and contradictory. In our work, real basal (hypothetical, corrected to the zero concentration of atmospheric oxygen), basal (atmospheric oxygen-stimulated) and stimulated (L-glutamic and hydrochloric acids) levels of TBA-reactive products (TBARP) were estimated in the brain (hippocampus--HPC, cortex--COR, cerebellum--CRB) from young (3 months) and old (28 months) male and female Wistar rats. The values of basal levels of TBARP were different in young (HPC > COR > CRB) as well as in old animals (COR > CRB > HPC). Thus, the process of aging caused a significant decrease in the HPC (to 72%) and no change in the COR and CRB. Levels stimulated by L-glutamic acid were significantly decreased (COR, CRB) and unchanged (HPC) in old compared to young animals. The real basal levels estimated by regression analysis seem to be higher in the brain tissue damaged by aging. We discuss the usefulness of the TBA test applied to aged tissue, the effect of acidosis, the effectivity of L-glutamic acid to generate free radicals and the differences between individual brain areas.

Aging↗
PubMedDate not supplied

[Toxicological characteristics of disodium salt of N-lauroyl-L-glutamic acid--a surface-active agent used in the food processing industry].

To synthesize the disodium salt of N-lauroyl-L-glutamic acid (LG-Na2), use was made of the chloranhydride method with some modifications. The yield of the product was 85%. LG-Na2 possesses good surface-active properties and an emulsifying effect. The critical concentration of micelloformation of LG-Na2 is 6 10(-3) mol/l. LG-Na2 is a non-toxic compound. It is not cumulated in the animals' body. The LD50 for albino rats per os exceeds 10 000 mg/kg, that for mice 6500 mg/kg. It was shown in subacute experiments in rats that LG-Na2 did not produce any substantial influence on the animals' status or on some biochemical and morphological indicators of blood and organs. The authors suggest a tentative level of a safe oral action of LG-Na2 on man (10 mg per kg bw/day). LG-Na2 is likely to be widely used in food industry.

Adrenal Glands↗
PubMed1996

Glutamic acid decarboxylase in rat olfactory bulb: effect of ovarian steroids or male pheromones.

The effect of ovarian steroids and pheromones on the activity of glutamic acid decarboxylase, the enzyme that synthesizes gamma-aminobutyric acid (GABA), was studied in the rat olfactory bulbs. The enzyme activity was measured in the main and accessory olfactory bulbs at 11:00 h and 17:00 h in ovariectomized rats, and in rats treated with ovarian steroids or exposed to male pheromones. The enzyme activity in both bulbs showed a diurnal fluctuation that was not affected in the accessory bulbs by the exposure to pheromones while the rhythm disappeared in the main bulbs. Estrogen and estrogen-progesterone treatments decreased the enzyme activity in both bulbs either in the morning or in the afternoon. The exposure of ovariectomized estrogen-primed rats to male pheromones reversed the effect of estrogen on the enzyme activity in the morning but not in the afternoon. Ovarian hormones plus pheromones prevented the steroid effect only in the morning. These results support the view that in olfactory bulbs, the GABAergic system can be modulated by endocrine and pheromonal factors.

Animals↗
PubMed1996

Increased thermo-stability of rat prolactin after replacing glutamic acid at position 118 by lysine.

We examined the structural stabilities after heat treatment of 22 mutants of rat prolactin (rPRL) with amino acid replacements at 15 different positions and recombinant wild-type rPRL (WT-PRL) as part of our series of studies on site-directed mutagenesis of rPRL. When WT-PRL at low concentrations (0.1-10 ng/ml) was heated at 100 degrees C for 20 min, it lost its Nb2 proliferation activity, whereas at high concentrations (above 1 microgram/ml), its activity remained. Temperature-dependent loss of the proliferation activity of 10 ng/ml WT-PRL after heat treatment for 5 min was observed. Next, we examined the proliferation activities of the 22 mutants heated at 60 and 70 degrees C for 5 min. After treatment at 60 degrees C, all the mutants retained their initial proliferation activities, whereas treatment at 70 degrees C reduced their activities to about 63%, except for one in which glutamic acid at position 118 was replaced by lysine (E118K), suggesting that the mutations did not induce structural instability. The mutant E118K retained 84% of its initial activity after treatment at 70 degrees C, significantly (P < 0.01) higher than the WT-PRL value. The temperature-dependency profile of the Nb2 proliferation activity of E118K also showed it had significantly increased thermo-stability. Meanwhile another mutant (E118Q) at the same residue showed no increased thermo-stability, suggesting that changing a negative charge (E) to a positive one (K) at position 118 induces ionic bond formation with a neighboring negative charge, resulting in thermostabilization of the structure of PRL.

Animals↗
PubMed2004

Glutamic acid decarboxylase autoimmunity in Batten disease and other disorders.

Degenerative diseases of the CNS, such as stiff-person syndrome (SPS), progressive cerebellar ataxia, and Rasmussen encephalitis, have been characterized by the presence of autoantibodies. Recent findings in individuals with Batten disease and in animal models for the disorder indicate that this condition may be associated with autoantibodies against glutamic acid decarboxylase (GAD), an enzyme that converts the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Anti-GAD autoantibodies could result in excess excitatory neurotransmitters, leading to the seizures and other symptoms observed in patients with Batten disease. The pathogenic potential of GAD autoantibodies is examined in light of what is known for other autoimmune disorders, such as multiple sclerosis, SPS, Rasmussen encephalitis, and type 1 diabetes, and may have radical implications for diagnosis and management of Batten disease.

Animals↗
PubMed2003

Distribution of glycine transporter 2 mRNA-containing neurons in relation to glutamic acid decarboxylase mRNA-containing neurons in rat medulla.

We studied the distribution of medullary glycinergic neurons in relation to GABAergic neurons, by using in situ hybridization method for mRNA encoding either glycine transporter 2 (GLYT2) or glutamic acid decarboxylase isoform 67 (GAD67). GLYT2 mRNA-positive (GLYT2+) neurons were distributed widely and clustered in (1). the respiration-related area of the ventrolateral medulla called the Bötzinger complex, (2). the nucleus retroambiguus caudal to the obex or the caudal ventral respiratory group, (3). the spinal trigeminal nucleus, (4). a small area immediately dorsal to the inferior olivary nucleus, and (5). the border zone between the hypoglossal nucleus and the surrounding reticular formation. It was characteristic that in the dorsomedial medulla, GLYT2+ neurons were distributed only sparsely in contrast to dense GAD67+ neurons. Only few GLYT2+ neurons were distributed in the medial and interstitial subnuclei of the nucleus tractus solitarii. In particular virtually no GLYT2+ neurons were found in the area postrema. Furthermore, in the reticular formation and the spinal trigeminal nucleus, GAG67+ neurons tended to be distributed in the area where GLYT2+ neurons were sparse, and vice versa. These results provide useful information for the effort of determining neurotransmitters involved in the medullary neurons.

Amino Acid Transport Systems, Neutral↗
PubMed1996

Allele-specific motifs characterize HLA-DQ interactions with a diabetes-associated peptide derived from glutamic acid decarboxylase.

Polymorphic residues of HLA class II molecules influence immune activation in part by determining specific structural constraints for binding antigenic peptides. We identified a peptide from glutamic acid decarboxylase, a diabetes-associated autoantigen that preferentially bound to HLA-DQ3.2 molecules, one of the HLA determinants highly associated with insulin-dependent diabetes. We analyzed interactions of specific HLA-DQ residues with modified peptide analogues and found a pattern of permissive site-specific amino acids that accommodated allele-specific binding. Four anchor residues constrain binding to different DQ alleles; limited variation at two of these sites, residues 4 and 9, accounts for the unique pattern of peptide binding to HLA-DQ3.1 or HLA-DQ3.2.

Alleles↗
PubMed1986

Effect of warfarin sodium therapy on excretion of 4-carboxy-L-glutamic acid in scleroderma, dermatomyositis, and myositis ossificans progressiva.

The effect of warfarin sodium on excretion of calcium, phosphorus, and 4-carboxy-L-glutamic acid (Gla) was studied in 5 patients with ectopic calcification (2 with scleroderma, 1 with dermatomyositis, and 2 with myositis ossificans progressiva). Warfarin reduced urinary excretion of Gla in all patients, but no changes in calcium and phosphorus excretion or in objective parameters of calcinosis were observed during 6-36 months of treatment. Two patients experienced hemorrhagic complications during therapy, emphasizing a hazard of long-term anticoagulation treatment. Since ectopic calcium deposits contain Gla-rich protein, suppression of Gla synthesis by warfarin sodium over a longer period may prevent deposition and allow removal of existing calcinosis deposits.

Adult↗
PubMed1995

Glutamic acid 327 in the sheep alpha 1 isoform of Na+,K(+)-ATPase is a pivotal residue for cation-induced conformational changes.

The cation binding characteristics of the mutant E327A formed in the sheep alpha 1 isoform of the Na+,K(+)-ATPase were examined using [3H]ouabain binding as a function of monovalent cation concentrations. Equilibrium competition binding assays in the presence of Mg2+, inorganic phosphate and various amounts of unlabelled ouabain indicated that both wild-type sheep alpha 1 protein and the E327A mutant expressed in 3T3 cells had similar affinities for ouabain (KD = 1.53 and 1.31 nM respectively). Sodium inhibition of ouabain binding appeared competitive in both enzymes. However, binding of three Na+ ions was required to explain the steep character of the Na+ inhibition curve for the wild-type Na+,K(+)-ATPase (Ki = 12.8 +/- 1.6 mM), whereas the binding of two Na+ ions was detected for the mutant E327A (Ki = 19.2 +/- 2.5 mM). Potassium binding of [3H]ouabain binding displayed a partially competitive nature with Hill coefficients of 2 for both wild-type sheep alpha 1 (Ki = 0.743 +/- 0.044 mM) and E327A (Ki = 0.875 +/- 0.067 mM). At concentrations of K+ above 10 mM, the sheep alpha 1 competition curve levelled off whereas the inhibition curve for E327A displayed a stimulation in ouabain binding. This stimulation in [3H]ouabain binding also occurred with Rb+, Cs+ and Li+, but was never observed with choline or Na+, suggesting that this effect was not due to ionic strength. From these [3H]ouabain-binding studies, it is obvious that the mutant enzyme E327A in the presence of Mg2+, Pi and ouabain, interacts with monovalent cations in a unique fashion. One interpretation of these data is that the glutamic acid residue at position 327 is involved in a conformational transition induced by the binding of monovalent cations to the Na+,K+-ATPase and that this transition is inhibited by the mutation of E327A.

3T3 Cells↗