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A J Tobin

Publications and source records attributed to A J Tobin.

At least 55 records · Page 3Linked to original sources
PubMed1993

Rat-1 fibroblasts engineered with GAD65 and GAD67 cDNAs in retroviral vectors produce and release GABA.

We have used a retroviral cDNA expression system to drive the expression of the different forms of glutamic acid decarboxylase (GAD65, GAD67, or both). Individual clones of engineered Rat-1 cells make the appropriate GAD mRNAs and GAD polypeptides, show GAD enzymatic activity, and make GABA. Clones expressing GAD65 had higher enzymatic activity than those expressing GAD67. As is the case for brain GADs and for GADs produced in engineered bacteria, the enzymatic activity of GAD65 is more responsive to added pyridoxal phosphate than that of GAD67. Immunostaining for both GADs is scattered throughout the cytoplasm. GAD65 immunostaining is less homogeneous than that of GAD67 and also appears to be associated with the surfaces of large vesicle-like structures. Cells expressing GAD65 and GAD67 showed similar immunostaining patterns with anti-GABA antibodies and contained substantial amounts of GABA (ranging from 7 to 18 pmol of GABA/10(6) cells), which was roughly proportional to their levels of GAD activity. GABA is released from the engineered cells into the surrounding medium under resting conditions, suggesting that cells programmed with GAD cDNAs might serve as effective sources of GABA in cell transplantation experiments.

Animals↗
PubMed1993

GABA alters GABAA receptor mRNAs and increases ligand binding.

In addition to its role as an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) influences the cytodifferentiation of developing neurons both in culture and in vivo. Here, we report some of the targets of GABA action and the mechanism through which GABA acts. In primary cultures of cerebellar granule cells, GABA specifically stimulates an increase in the levels of mRNAs for alpha 1 and beta 2 GABAA receptor subunits. The GABAA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) mimics this effect, and the GABAA antagonist bicuculline prevents it. In addition, GABA and THIP trigger an increase in the number of GABA binding sites. This increase parallels that seen in vivo, where the total number of GABAA receptor sites increases during postnatal cerebellar development. It is interesting that the period of the greatest increase in the number of receptor sites coincides with the development of the granule cells. Taken together, our data suggest that GABA may play an important role during maturation of cerebellar granule cells by influencing the number and composition of its own receptors.

Animals↗
PubMed1993

Islet cell cytoplasmic autoantibody reactivity to glutamate decarboxylase in insulin-dependent diabetes.

Individuals with or at risk for insulin-dependent diabetes (IDD) frequently have autoantibodies against an islet cell cytoplasmic (ICA) antigen thought to be a sialoglycolipid. However, we now report that preabsorption of ICA-positive sera with recombinant glutamate decarboxylase (human GAD 65 and/or GAD 67) reduced or blocked the ICA reactivity of 5/18 (27%) new-onset IDD patients and 7/18 (39%) prediabetics. Interestingly, nondiabetic subjects with ICA of > or = 5 yr in duration had GAD-reactive ICA significantly more often (16/24, 67%, P < 0.04) than the diabetic groups. ICA reactivity to GAD was not related to serum ICA titer nor the age of the individual, and in all cases tested was blocked by GAD 65 or GAD 67 with equivalent efficiency. The ICA observed in 21/25 (84%) IDD patients with ICA long after clinical onset of disease (9-42 yr) was reactive to GAD. A natural history analysis of three individuals showed conversions from ICA which was reactive to GAD to a non-GAD-reactive ICA nearer to their clinical onsets of IDD. This study further defines the autoantigens reactive to ICA, and suggests that, whereas ICA that are not reactive to GAD may identify an advanced and more prognostic lesion, GAD-reactive ICA may typify the early or inductive lesion that may or may not progress to clinically significant beta cell injury.

Adolescent↗
PubMed1992

Expression of GABAA receptor polypeptides in clonal rat cell lines.

The neuronal-like cell lines, B35, B65, B103, and B104, previously reported to possess high affinity GABA binding, were analyzed for various cellular properties. They possessed peripheral but lacked central benzodiazepine binding. Only B65 cells possessed [3H-]muscimol binding; none bound [35S]TBPS. None of the cells exhibited GABA-stimulated chloride conductance with patch clamp recordings. By Western blots the cells possessed alpha subunits. Northern blot and polymerase chain reaction analysis showed that out of alpha 1, alpha 4, beta 1 and gamma 2 subunits, only alpha 1 subunit mRNA was present. Thus, GABAA-receptor binding without associated central benzodiazepine receptor sites and without functional chloride channels appears to result from expression of an incomplete subunit composition.

Animals↗
PubMed1992

Isolation and cloning of a voltage-dependent anion channel-like Mr 36,000 polypeptide from mammalian brain.

A polypeptide of M(r) 36,000 (36 kDa) was isolated from detergent-solubilized membrane fractions of mammalian brain on a benzodiazepine affinity column utilized for the purification of the gamma-aminobutyric acid/benzodiazepine receptor protein, followed by preparative gel electrophoresis. Partial protein sequence for two fragments of the 36-kDa polypeptide allowed the isolation of cDNA clones from a rat hippocampal library. An open reading frame coding a sequence of 295 amino acid residues containing the two probe peptide sequences with minor differences, and a putative N-terminal signal peptide of 25 residues was found. Hydropathy index revealed no regions of alpha-helix suitable for membrane spanning, but several areas of alternating hydrophilic and hydrophobic residues consistent with beta-strands. The sequence of this brain protein was 24% identical to that of a yeast mitochondrial protein, the voltage-dependent anion channel (VDAC), and over 70% identical with the VDAC from human B lymphocytes. The gamma-aminobutyric acid type A (GABAA) receptor/36-kDa preparation purified on benzodiazepine affinity column has channel-forming activity in lipid bilayer membranes that is virtually identical to VDAC isolated from mitochondria of various sources, indicating that the 36-kDa protein is a new member of the VDAC family of proteins. An antiserum raised against the purified 36-kDa polypeptide was able to precipitate [3H]muscimol binding activity, indicating a tight association with the GABAA receptor protein in vitro and copurification on the benzodiazepine affinity column due to this association. Further studies are needed to determine whether such an association occurs in vivo.

Amino Acid Sequence↗
PubMed1992

Comparative distribution of messenger RNAs encoding glutamic acid decarboxylases (Mr 65,000 and Mr 67,000) in the basal ganglia of the rat.

Glutamic acid decarboxylase, the enzyme required for GABA synthesis, exists as distinct isoforms, which have recently been found to be encoded by different genes. The relative expression of messenger RNAs encoding two isoforms of glutamic acid decarboxylase (Mr 67,000 and Mr 65,000) was measured at the single-cell level in neurons of the rat basal ganglia with in situ hybridization histochemistry. Both messenger RNAs were expressed in neurons of the striatum, pallidum, and substantia nigra pars reticulata, but marked differences in the relative level of labelling were observed with the two probes. In striatum, efferent neurons were more densely labelled for the messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) than for the messenger RNA encoding glutamic acid decarboxylase (Mr 67,000), whereas the reverse was observed for GABA-ergic interneurons. Neurons of the entopeduncular nucleus were much more densely labelled for messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) than for messenger RNA encoding glutamic acid decarboxylase (Mr 67,000). In addition, labelling for messenger RNA encoding glutamic acid decarboxylase (Mr 65,000) was higher in the entopeduncular nucleus (internal pallidum) than in the globus pallidus (external pallidum), a structure which expressed similar levels of both mRNAs. In contrast to neurons of the internal pallidum, efferent neurons of the substantia nigra pars reticulata expressed slightly more messenger RNA encoding glutamic acid decarboxylase (Mr 67,000) than that encoding the other isoform of the enzyme. The results suggest a differential expression of the messenger RNAs encoding the two isoforms of glutamic acid decarboxylase in subpopulations of basal ganglia neurons in rats.

Animals↗
PubMed1992

Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene.

We report the isolation and sequencing of cDNAs encoding two human glutamate decarboxylases (GADs; L-glutamate 1-carboxy-lyase, EC 4.1.1.15), GAD65 and GAD67. Human GAD65 cDNA encodes a Mr 65,000 polypeptide, with 585 amino acid residues, whereas human GAD67 encodes a Mr 67,000 polypeptide, with 594 amino acid residues. Both cDNAs direct the synthesis of enzymatically active GADs in bacterial expression systems. Each cDNA hybridizes to a single species of brain mRNA and to a specific set of restriction fragments in human genomic DNA. In situ hybridization of fluorescently labeled GAD probes to human chromosomes localizes the human GAD65 gene to chromosome 10p11.23 and the human GAD67 gene to chromosome 2q31. We conclude that GAD65 and GAD67 each derive from a single separate gene. The cDNAs we describe should allow the bacterial production of test antigens for the diagnosis and prediction of insulin-dependent diabetes mellitus.

Amino Acid Sequence↗
PubMed1992

Response of peripheral-blood mononuclear cells to glutamate decarboxylase in insulin-dependent diabetes.

Insulin-dependent diabetes is characterised by autoantibodies to several pancreatic-islet-cell antigens, including glutamate decarboxylase. We measured the proliferative responses to this antigen of peripheral-blood mononuclear cells from patients with newly diagnosed insulin-dependent diabetes, relatives of diabetic patients, and healthy controls. The likelihood of a positive response was substantially greater among the diabetic patients and relatives positive for islet-cell autoantibodies (ICA) than among subjects at low risk of diabetes (controls and ICA-negative relatives). Glutamate decarboxylase may have a pathogenetic role in insulin-dependent diabetes.

Autoantibodies↗
PubMed1992

Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.

Animals↗
PubMed1992

Glutamate decarboxylases in nonneural cells of rat testis and oviduct: differential expression of GAD65 and GAD67.

gamma-Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5'-phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.

Animals↗
PubMed1992

A transcriptional regulatory element of the gene encoding the 67,000-M(r) form of human glutamate decarboxylase is similar to a Drosophila regulatory element.

We have isolated the 5' flanking DNA sequences of the human gene encoding the 67,000-M(r) form of glutamate decarboxylase (GAD67), the gamma-aminobutyric acid synthetic enzyme. Transcription begins at a single promoter (P1) in adult brain but at two tandem promoters, P1 and P2, in fetal brain. P1, which is 3' to P2, resembles the promoter regions of many constitutively expressed genes, whereas P2 resembles a tissue-specific promoter. P1 contains a 10-base sequence (dec-1) that closely matches the element I cis-regulatory sequence identified in the promoter region of Drosophila 3,4-dihydroxyphenylalanine decarboxylase. Gel shift and transient expression assays demonstrate that the dec-1 sequence plays a role in the transcription of the human GAD67 gene.

Animals↗
PubMed1992

Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is thought to result from the autoimmune destruction of the insulin-producing beta cells of the pancreas. Years before IDDM symptoms appear, we can detect autoantibodies to one or both forms of glutamate decarboxylase (GAD65 and GAD67), synthesized from their respective cDNAs in a bacterial expression system. Individual IDDM sera show distinctive profiles of epitope recognition, suggesting different humoral immune responses. Although the level of GAD autoantibodies generally decline after IDDM onset, patients with IDDM-associated neuropathies have high levels of antibodies to GAD, years after the appearance of clinical IDDM. We note a striking sequence similarity between the two GADs and Coxsackievirus, a virus that has been associated with IDDM both in humans and in experimental animals. This similarity suggests that molecular mimicry may play a role in the pathogenesis of IDDM.

Amino Acid Sequence↗
PubMed1992

Immortalized hypothalamic GT1-7 neurons express functional gamma-aminobutyric acid type A receptors.

Neuronal cell lines provide a source of pure populations of neurons and allow the properties of many neurotransmitter receptors to be studied. However, none of these cells have been reported to express functional gamma-aminobutyric acid (GABA)A receptors. Indeed, there have been no reports of cell lines expressing functional amino acid receptors. Using biochemical and electrophysiological techniques, we have identified a neuronal cell line expressing functional GABAA receptors. Membranes from immortalized hypothalamic (GT1-7) neurons bound [3H]muscimol but not [3H]flunitrazepam. GABA-activated chloride currents, recorded from GT1-7 cells, were blocked by bicuculline and Zn2+ but were insensitive to diazepam. These results suggest that GABAA receptors on GT1-7 cells lack gamma subunits. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one and pentobarbital both modulated GABAA receptors in these cells. Polymerase chain reaction analysis of the cells revealed the presence of mRNAs encoding alpha 1, beta 1, and beta 3 polypeptides. GT1-7 cells provide a useful model system for studying the regulation of GABAA receptor polypeptide expression.

Anesthetics↗