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PubMed1993

Transcriptional organization of a Drosophila glutamic acid decarboxylase gene.

We previously described the sequence and expression pattern of a Drosophila mRMA (Gad) that encodes the major soluble form of glutamic acid decarboxylase (GAD). We now report the transcriptional organization of the Drosophila Gad gene. Based on a combination of DNA sequence, RNase protection, primer extension, and polymerase chain reaction analyses, we conclude that the transcription unit for a 3.1-kb Gad mRNA is composed of eight exons that span approximately 17-kb genomic interval. By this analysis, the site of Gad transcript initiation overlaps with a recognition sequence that confers binding of the zeste transcription factor to other promoter elements. We emphasize that our analysis of the Gad transcription unit provides no evidence for alternative RNA splicing as a mechanism for the generation of GAD isoforms. Thus, the several GAD-immunoreactive proteins (putative GAD isoforms) that can be detected in Drosophila extracts are probably encoded by distinct genes.

Animals↗
PubMed1999

Inhibition of mitochondrial oxidative phosphorylation induces hyper-expression of glutamic acid decarboxylase in pancreatic islet cells.

It has been hypothesised that mitochondrial dysfunction in pancreatic beta cells could produce hyper-expression of glutamic acid decarboxylase (GAD), a major autoantigen in insulin-dependent diabetes mellitus (IDDM) (Degli Esposti, M. and Mackay, I.R. Diabetologia 40: 352-356, 1997). Here we report that specific inhibition of mitochondrial respiration enhances the expression of GAD in both foetal mouse pancreatic tissue and hamster HIT-T15 cells. Inhibitors of NADH-ubiquinone oxidoreductase (complex I) seem to be particularly effective in increasing the expression of GAD in both foetal mouse pancreas and HIT-T15 hamster beta cells, especially in the presence of nutrients such as arginine and glucose. These results represent the first evidence that GAD expression is enhanced under conditions that are toxic to pancreatic beta cells, and establish a link between mitochondrial dysfunction and expression of IDDM autoantigens.

Animals↗
PubMedDate not supplied

Simultaneous demonstration of choline acetyltransferase and glutamic acid decarboxylase immunoreactivity in the rat interpeduncular nucleus.

Double antigen immunohistochemistry was employed to simultaneously examine the distribution of choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) immunoreactivity in the rat interpeduncular nucleus (IPN) at the subnuclear and ultrastructural level. ChAT-immunoreactive axons of the fasciculus retroflexus (FR) innervated specific subnuclear divisions of the IPN that possessed GAD-immunoreactive somata and a high density of GAD-immunoreactive axons and terminals. At the ultrastructural level, each of the cholinoceptive subnuclei possessed a characteristic axodendritic synaptic contact. These morphologically distinct synapses were composed of terminals of ChAT-positive FR axons forming asymmetric contacts with dendritic profiles of GAD-positive neurons. An array of symmetric axodendritic contacts with GAD immunoreactivity located pre- and/or postsynaptically was also present in the cholinoceptive subnuclear divisions. The present study provides direct evidence for synaptic interactions between ChAT-immunoreactive FR axons and dendritic processes of GAD-immunoreactive neurons in the rat IPN. Also, GAD-positive terminals arising from possible intrinsic projections contact dendritic profiles of GAD-immunoreactive neurons in receipt of ChAT-positive FR terminals. These results reveal that putative cholinergic afferent inputs and GABAergic intranuclear projections simultaneously innervate a subpopulation of IPN neurons that possess GAD immunoreactivity.

Animals↗
PubMed1977

The apparent absence of involvement of biotin in the vitamin K-dependent carboxylation of glutamic acid residues of proteins.

The mechanism of the vitamin K-dependent post-translational carboxylation of the gamma-carbon atom of glutamic acid residues in proteins remains obscure. Experiments were performed in vivo and in vitro in an attempt to establish a role for biotin in the transfer of the carboxyl group. Weanling male rats were fed on a biotin-deficient diet until severe biotin deficiency was induced. Their degree of biotin deficiency was documented by assaying for liver acetyl-CoA carboxylase activity, which was about 15% of normal. However, one-stage and two-stage prothrombin times measured on the plasmas were normal. In addition, the liver microsomal fraction did not contain any more prothrombin precursor than did that of normal rat liver. Experiments were done in vitro in which vitamin K-dependent fixing of 14CO2 was measured in the liver microsomal fraction from vitamin K-deficient male rats in the presence or absence of avidin. No evidence for an avidin-sensitive critical biotin-containing site was obtained. Thus neither series of experiments suggests a role for biotin; the data are compatible with carboxyl transfer occurring either through a carboxylated vitamin K intermediate; or via a yet to be identified intermediate, or perhaps via CO2 itself.

Acetyl-CoA Carboxylase↗
PubMed1983

Immunocytochemical localization of glutamic acid decarboxylase in normal and deafferented superior colliculus: evidence for reorganization of gamma-aminobutyric acid synapses.

There is circumstantial evidence that GABAergic synaptic terminals in the superior colliculus might become reorganized in response to a loss of the retinal innervation of this brain region. The present investigation tests this possibility by identifying gamma-aminobutyric acid (GABA) neurons and their synaptic relationships with an immunocytochemical localization of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), and by comparing these synaptic relationships in normal superior colliculus with those present 6 and 16 weeks after unilateral eye removal. In normal superior colliculus, light microscopy revealed a much denser concentration of GAD-positive reaction product in the superficial layers than in the intermediate and deep collicular layers. Most of this reaction product was contained within small, punctate structures, but GAD-positive somata and proximal dendrites also were observed. Electron microscopy showed that GAD was localized in numerous synaptic terminals, including those that were the presynaptic elements of dendrodendritic synapses. The vast majority of GAD-positive presynaptic elements formed symmetric synaptic junctions. In addition, GAD-positive profiles frequently were postsynaptic to unstained retinal terminals as well as presynaptic to unstained dendritic profiles and thus participated in serial synaptic relationships. Furthermore, both retinal and GAD-positive elements commonly were presynaptic to the same postsynaptic dendrite, and often these synaptic contracts were adjacent to each other. In deafferented specimens, profiles with the characteristics of retinal axon terminals were not observed, whereas there appeared to be no reduction of GAD-positive synaptic profiles. However, there was a marked and statistically significant increase in the proportion of GAD-positive presynaptic terminals that formed asymmetric synaptic contacts in superior colliculus deprived of retinal input. This change indicates that partial deafferentation induces a reorganization of GAD-positive synapses. The long-term presence of the GABA-synthesizing enzyme within reorganized synaptic terminals also suggests that such presynaptic elements could produce and, presumably, release neurotransmitter.

Animals↗
PubMed2003

Regulatory cytokine production stimulated by DNA vaccination against an altered form of glutamic acid decarboxylase 65 in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop a T-cell dependent autoimmune form of diabetes, in which glutamic acid decarboxylase 65 (GAD65) is an important islet target antigen. Intramuscular DNA vaccination with a plasmid encoding native GAD65 (a cytosolic antigen) did not significantly alter the incidence of diabetes, but vaccination against an altered form of GAD65 with a signal peptide (spGAD), which is secreted in vitro, was protective. The preventive effect was further enhanced by repeated injections of the spGAD plasmid. Following DNA injection into muscle GAD65 was expressed for several months, and this was not accompanied by an inflammatory response. Immunization against GAD65 was not associated with substantial alterations in cytokine production by splenic lymphocytes stimulated with immunogenic GAD65 peptides. In contrast, spGAD induced increased secretion of both interleukin 10 and interferon gamma and a striking decrease in the interferon gamma/interleukin 10 ratio in culture supernatants. Similarly, spGAD-immunized mice had higher serum interleukin 10 levels and lower serum interferon gamma levels than other groups, suggesting a systemic effect. In nondiabetic mice there was increased basal production of transforming growth factor beta(1), which was enhanced by antigenic stimulation. These alterations in regulatory cytokine production were apparent both early and late after the treatment was initiated. These findings suggest that DNA vaccination against spGAD protects NOD mice by increasing regulatory cytokine production.

Animals↗
PubMed1993

Comparative localization of mRNAs encoding two forms of glutamic acid decarboxylase with nonradioactive in situ hybridization methods.

Nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes were used to localize two glutamic acid decarboxylase (GAD) mRNAs in rat brain. These mRNAs encode two forms of GAD that both synthesize GABA but differ in a number of characteristics including their molecular size (65 and 67 kDa). For each GAD mRNA, discrete neuronal labeling with high cellular resolution and low background staining was obtained in most populations of known GABA neurons. In addition, the current methods revealed differences in the intensity of labeling among neurons for each GAD mRNA, suggesting that the relative concentrations of each GAD mRNA may be higher in some groups of GABA neurons than in others. Most major classes of GABA neurons were labeled for each GAD mRNA. In some groups of GABA neurons, the labeling for the two mRNAs was virtually identical, as in the reticular nucleus of the thalamus. In other groups of neurons, although there was substantial labeling for each GAD mRNA, labeling for one of the mRNAs was noticeably stronger than for the other. In most brain regions, such as the cerebellar cortex, labeling for GAD67 mRNA was stronger than for GAD65 mRNA, but there were a few brain regions in which labeling for GAD65 mRNA was more pronounced, and these included some regions of the hypothalamus. Finally, some groups of GABA neurons were predominantly labeled for one of the GAD mRNAs and showed little or no detectable labeling for the other GAD mRNA, as, for example, in neurons of the tuberomammillary nucleus of the hypothalamus where labeling for GAD67 mRNA was very strong but no labeling for GAD65 mRNA was evident. The findings suggest that most classes of GABA neurons in the central nervous system (CNS) contain mRNAs for at least two forms of GAD, and thus, have dual enzyme systems for the synthesis of GABA. Higher levels of one or the other GAD mRNA in certain groups of GABA neurons may be related to differences in the functional properties of these neurons and their means of regulating GABA synthesis.

Animals↗
PubMed2004

Presence of diabetes-inhibiting, glutamic acid decarboxylase-specific, IL-10-dependent, regulatory T cells in naive nonobese diabetic mice.

Immunization of NOD mice with autoantigens such as glutamic acid decarboxylase (GAD) 221-235 peptide (p221) can induce Ag-specific CD4(+) T regulatory (Tr) cells. However, it is unclear whether these Tr cells acquire their regulatory capacity due to immunization or whether they are constitutively harbored in unimmunized naive mice. To address this question, we used an I-Ag7 tetramer to isolate p221-specific T cells from naive NOD mice (N221(+) cells) after peptide-specific in vitro expansion. The N221(+) T cells produced IFN-gamma and IL-10, but very little IL-4, in response to p221 stimulation. These T cells could function as regulatory cells and inhibit in vitro proliferation of diabetogenic BDC2.5 cells. This suppressive activity was cell contact-independent and was abrogated by Abs to IL-10 or IL-10R. Interestingly, IL-2 produced by other T cells present in the cell culture induced unactivated N221(+) T cells to exhibit regulatory activities involving production of IL-10. In vivo, N221(+) cells inhibited diabetes development when cotransferred with NOD splenocytes into NOD/scid recipients. Together, these results demonstrate that p221-specific IL-10-dependent Tr cells, including Tr type 1 cells, are present in naive NOD mice. The use of spontaneously arising populations of GAD peptide-specific Tr cells may represent a promising immunotherapeutic approach for preventing type 1 diabetes.

Adoptive Transfer↗
PubMed1981

Possible role of prolactin in the modification of medial basal hypothalamic glutamic acid decarboxylase activity.

The effects of acute and chronic injections of haloperidol, sulpiride and apomorphine on serum prolactin (PRL) levels and medial basal hypothalamus (MBH) glutamic acid decarboxylase (GAD) activity were investigated in male rats. Parallel changes in PRL and GAD activity were observed in acutely treated animals. Conversely, a return to normal of the GAD activity associated with high plasma PRL levels was induced by chronic haloperidol and sulpiride treatment. Results are discussed in the light of the possible existence of a hypothetical PRL-gamma-aminobutyric acid (GABA) subsidiary feedback loop.

Animals↗
PubMed2000

Anti-sense oligonucleotides, for progestin receptors in the VMH and glutamic acid decarboxylase in the VTA, attenuate progesterone-induced lordosis in hamsters and rats.

Immunocytochemical (ICC) staining for progesterone (P) receptors (PRs) and glutamic acid decarboxylase (GAD), the enzyme responsible for GABA production, reveal that there are many PRs in the ventral medial hypothalamus (VMH) and many GAD containing neurons in the ventral tegmental area (VTA). To investigate P's action on lordosis in the VMH and VTA, anti-sense oligos specific to PRs and GAD(65&67) were intracerebrally infused into the VMH and VTA of 24 ovariectomized hamsters and 40 ovariectomized rats. Estradiol benzoate (2 microg) primed hamsters and rats were infused to the VMH and the VTA with either PR (250 ng/1.0 microl infusion) or GAD (500 ng/1.0 microl infusion) anti-sense oligos, their scramble controls, or saline vehicle at hour 0 and again at hour 24. At hr 44, rodents were subcutaneously injected with P (500 microg) and were tested for sexual receptivity with a male 4 h later. There were significant reductions in lordosis of hamsters and rats following PR anti-sense infusions to the VMH compared to scrambled or vehicle control infusions. Effects of PR anti-sense to the VMH were not different from combined VMH and VTA PR anti-sense infusions; however, VMH infusions reduced lordosis compared to VTA-only anti-sense infusions. GAD anti-sense infusions reduced lordosis when infused into the VTA, compared to scrambled or saline vehicle infusions. Lordosis responsiveness following VTA GAD anti-sense infusions was not different from combined VMH and VTA infusions, but VTA infusions of GAD anti-sense reduced lordosis compared to VMH-only anti-sense infusions. These data suggest that in the VMH, PRs are important for P-facilitated lordosis, whereas in the VTA, GABAergic neurons may be an important substrate for mediating P's actions on lordosis of rodents.

Animals↗
PubMed1986

Immunoreactive glutamic acid decarboxylase in the trigeminal nucleus caudalis of the cat: a light- and electron-microscopic analysis.

This study used antisera directed against glutamic acid decarboxylase (GAD), the biosynthetic enzyme for gamma-aminobutyric acid (GABA), to examine the light- and electron-microscopic distribution of presumed GABA-ergic synapses in the medullary homologue of the cat spinal dorsal horn, the trigeminal nucleus caudalis. At the light-microscopic level, immunoreactive terminals were concentrated in the superficial dorsal horn, laminae I and II. Colchicine was generally ineffective in revealing the distribution of cell bodies. However, in two successful cases, the majority of labeled cells were found in the magnocellular layer, ventral to the substantia gelatinosa, a region that had a lower density of immunoreactive terminals. Other labeled neurons were scattered in laminae I and II. A variety of synaptic arrangements were found at the electron-microscopic level. These derived from two types of labeled terminals. One contained both small round vesicles and large dense-cored vesicles. The second contained small round and pleomorphic vesicles. Some immunoreactive GAD terminals contained a few flat vesicles. Labeled terminals predominantly formed axodendritic synapses, via symmetrical contacts. Several axoaxonic arrangements were also observed. In most cases, the GAD terminal (which did not contain dense-cored vesicles) was presynaptic to another vesicle-containing profile, including the scalloped central terminal thought to derive from primary afferents. Another population of labeled GAD terminals was found postsynaptic to unlabeled vesicle-containing profiles, including central terminals. These data indicate that inhibitory GABA-ergic controls in the trigeminal nucleus caudalis involve both presynaptic and postsynaptic mechanisms and are probably mediated via direct contacts onto ascending projection neurons, as well as via synaptic contacts onto nociceptive primary afferent fibers. The transmission of nociceptive messages by neurons of the spinal cord dorsal horn and trigeminal nucleus caudalis is subject to a variety of segmental and supraspinal controls. Pharmacological and electrophysiological studies have implicated the biogenic amines serotonin and norepinephrine, and the endogenous opioid peptides enkephalin and dynorphin, in those controls (Basbaum and Fields, 1978, 1984; Basbaum et al., 1983; Basbaum, 1985).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2000

Thyroid autoantibodies in Thai type 1 diabetic patients: clinical significance and their relationship with glutamic acid decarboxylase antibodies.

OBJECTIVE: To study the clinical significance of thyroid autoantibodies in Thai patients with type 1 diabetes and their relationship with glutamic acid decarboxylase antibodies (GAD(65)Ab). METHODS: Thyroglobulin antibodies (TG-Ab) and thyroid peroxidase antibodies (TPO-Ab) were measured in 50 Thai type 1 diabetic patients. Forty-four patients also had GAD(65)Ab measured. Serum thyrotropin (TSH) was measured in all patients who had no history of thyroid disease regardless of thyroid antibody status. Clinical data including sex, age at onset and duration of diabetes, family history of diabetes, fasting c-peptide levels as well as frequencies of GAD(65)Ab were compared between patients with and without thyroid antibodies. GAD(65)Ab was also measured in 29 non-diabetic patients with hyperthyroid Graves' disease or Hashimoto thyroiditis as a control group. RESULTS: TG-Ab and TPO-Ab were positive in nine (18%) and 15 (30%) patients, respectively. Eight patients (16%) were positive for both antibodies. Two of 16 patients who were positive for TG-Ab or TPO-Ab had a previous history of hyperthyroidism prior to diabetes onset. Of the remainder, two were newly diagnosed with hyperthyroidism and one was found to have clinical hypothyroidism at the time of the study. None of 34 patients without thyroid antibodies had thyroid dysfunction. Eight patients with positive thyroid antibodies but without clinical thyroid dysfunction and 21 patients without thyroid antibodies were followed for up to 3 years, two patients of the first group developed hypothyroidism, whereas none of the latter developed thyroid dysfunction. The frequency of thyroid dysfunction at the time of initial study was significantly higher in patients with positive thyroid antibodies (3/14 vs. 0/34; P=0.021) and these patients who were initially euthyroid tended to have a higher risk of developing thyroid dysfunction (2/8 vs. 0/21; P=0.069). The frequency of thyroid antibodies was significantly increased in females and in those who had positive GAD(65)Ab. GAD(65)Ab was negative in all of the non-diabetic patients with autoimmune thyroid disease. CONCLUSIONS: About one-fourth of Thai patients with type 1 diabetes without thyroid disease had thyroid antibodies. The frequency of thyroid antibodies was increased in female and in GAD(65)Ab positive patients. The presence of thyroid antibodies is associated with a higher frequency of and may predict a higher risk for thyroid dysfunction in Thai type 1 diabetic patients.

Adult↗
PubMed1981

Immunotherapy with short ragweed fraction A:D-glutamic acid:D-lysine polymer in ragweed hay fever.

We report the first human trial of immunotherapy employing the nonimmunogenic carrier, D-glutamic acid:D-lysine linked to short ragweed (SRW) fraction A (fraction A:D-GL). Twelve SRW-sensitive patients with no immunotherapy during the previous 19 yr received a 2-mo (7/79 to 9/79) course of fraction A:D-GL (average dose 49.5 mg, range 21 to 78 mg). We compared their symptom scores and serologic changes with two control groups of SRW-sensitive patients. Patients receiving fraction A:D-GL demonstrated at least a tenfold decrease in skin-test sensitivity to SRW and had statistically lower mean seasonal symptom scores (p less than 0.02) than untreated controls. Mean seasonal symptom scored did not differ statistically from those of control patients on year 4 of immunotherapy. In contrast to the expected suppression of IgE, we found that fraction A:D-GL stimulated both IgE and IgG responses to SRW and SRW-antigen E. These increases in IgE and IgG antibodies were significantly greater than in the control groups and appeared to be due largely to injection of fraction A:D-GL. Though fraction A:D-GL was generally well tolerated, we noted mild generalized urticaria in three patients, and large local reactions in five others. The difference between our results and the earlier results in mice may reside in the particular characteristics of this preparation of fraction A:D-GL.

Adult↗
PubMed2000

Comparative expression and purification of human glutamic acid decarboxylase from Saccharomyces cerevisiae and Pichia pastoris.

The yeast cell factory is a potentially useful source of proteins in general. They include glutamic acid decarboxylase (GAD), which is one of the major autoantigens for Type 1 diabetes. We have created a hybrid form of GAD consisting of amino acids 1-101 of the human GAD67 protein fused to amino acids 96-585 of the human GAD65 protein, and have modified this to include a C-terminal hexa-Histidine (H6) tag sequence. This hybrid GAD67/65-H6 was expressed in two yeast hosts: constitutively under the control of the plasmid phosphoglycerate kinase promoter (PGK1) in Saccharomyces cerevisiae, and inducibly under the control of the chromosomal alcohol oxidase promoter (AOX1) in Pichia pastoris. Enzymatically active hybrid GAD was prepared from yeast lysates by purification either on an affinity column based on the GAD-1 monoclonal antibody, or by metal-affinity chromatography. The purified GAD67/65-H6 was radiolabelled with iodine-125 and tested with Type 1 diabetes sera in a radioimmunoprecipitation assay, and results were compared with those using untagged GAD67/65 and those using porcine brain GAD. The results of enzymatic and immunological assays show hybrid GAD67/65 is isolated at high specific activity and moderate yield, and the addition of the H6 tag sequences or the choice of yeast strain did not appreciably affect enzyme activity, percentage recovery of GAD, protein purification, or the utility in diagnosis of diabetes in terms of specificity and sensitivity to the various sera.

Journal Article↗