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PubMed1992

N-[4-[[(3,4-dihydro-4-oxo-1,2,3-benzotriazin-6- yl)methyl]amino]benzoyl]-L-glutamic acid, a novel A-ring analogue of 2-desamino-5,8-dideazafolic acid.

N-[4-[[(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-6- yl)methyl]amino]benzoyl]-L-glutamic acid ("2-aza-2-desamino-5,8- dideazafolic acid", ADDF) was synthesized from 2-amino-5-methylbenzamide via a four-step sequence consisting of diazotization, benzylic bromination, condensation with dimethyl N-(4-aminobenzoyl)-L-glutamate, and ester hydrolysis. ADDF was an inhibitor of recombinant mouse thymidylate synthase; inhibition was competitive with 5,10-methylenetetrahydrofolate as variable substrate (Ki = 2.3 microM). It was a substrate for murine folylpolyglutamate synthetase with kinetic characteristics (Km = 28 microM) comparable to those of aminopterin, and it inhibited the growth of L1210 cells in culture (IC50 = 0.52 microM). The structural modification of the A-ring embodied in ADDF appears to offer a novel, heretofore unexplored approach to the design of TS inhibitors.

Acyltransferases↗
PubMed1986

Distribution of glutamic acid decarboxylase-immunoreactive structures in the barrel region of mouse somatosensory cortex.

Neuronal structures in the barrel region of the mouse primary somatosensory cortex containing gamma-aminobutyric acid (GABA) were identified by an immunocytochemical method, using an antiserum to glutamic acid decarboxylase (GAD), the rate-limiting enzyme in the synthesis of GABA. GAD-positive cell bodies were found in all layers of the barrel cortex, but were more concentrated in the upper portion of layer II/III, and in layers IV and VI. Puncta, presumably axon terminals, were also distributed throughout the cortical layers; a high density of puncta occurs in layer IV, whereas a somewhat lower density characterizes layer VI. Based on the shapes of their somata and the distribution of their dendrites it was determined that all GAD-positive cell bodies were of the non-pyramidal type.

Animals↗
PubMed2004

Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein.

We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.

Autoantigens↗
PubMed1992

De novo mutation in the COL4A5 gene converting glycine 325 to glutamic acid in Alport syndrome.

Southern blot analysis of the COL4A5 gene in a 6 year old Italian Alport patient (proband VIZ) showed the loss of an MspI site that was present in the mother and control DNAs. PCR amplification and DNA sequencing revealed a single G-->A nucleotide change. The mutation results in substitution of a glutamic acid for a glycine residue at position 325 in the triple helical region of the alpha 5(IV) chain.

Base Sequence↗
PubMed1995

No association of antibodies to glutamic acid decarboxylase and diabetic complications in patients with IDDM.

OBJECTIVE: To evaluate the association of antibodies to glutamic acid decarboxylase (GAD-ab) and diabetic complications (neuropathy, retinopathy, and nephropathy) in patients with insulin-dependent diabetes mellitus (IDDM). RESEARCH DESIGN AND METHODS: We examined the prevalence of GAD-ab (immunoprecipitation assay) and islet cell antibodies (ICAs) (indirect immunofluorescence) in a representative sample of IDDM patients (n = 146) with different disease duration (2-52 years, median 13.2 years). Of all patients characterized for the existence of diabetic complications, 56 of 146 had peripheral neuropathy, 24 of 142 had autonomic neuropathy, 67 of 141 had retinopathy, and 39 of 146 had nephropathy. RESULTS: GAD-ab (> 2 SD) were detected more frequently than ICA (> 5 Juvenile Diabetes Foundation units) in IDDM patients of different disease duration (GAD-ab+ 37% [54 of 146] vs. ICA+ 22% [32 of 146], P = 0.011; diabetes duration less than median: GAD-ab+ 47% vs. ICA+ 23%, P = 0.0046; diabetes duration greater than median: GAD-ab+ 27% vs. ICA+ 22%, P > 0.05). For GAD-ab and for ICA, respectively, no difference was observed in frequency of positivity or titers between patients with or without diabetic complications. CONCLUSIONS: Both GAD-ab and, to a lesser extent, ICA persist for a long time in several individuals. This persistence is not related to diabetic neuropathy or any other diabetic complication.

Adolescent↗
PubMedDate not supplied

Distribution of glutamic acid decarboxylase messenger RNA-containing nerve cell populations of the male rat brain.

The distribution of glutamic acid decarboxylase (GAD) mRNA was investigated throughout the rat brain by means of in situ hybridization. Hybridization was carried out with a 35S-radiolabeled cRNA probe transcribed from a cDNA from cat occipital cortex and cloned in a SP6-T7 promoter-containing vector. Fixed tissue sections were hybridized with 35S GAD probe (0.6 kb length). Signal was detected by means of film or emulsion autoradiography. The autoradiograms were semiquantitatively evaluated by means of computer-assisted image analysis. The results obtained with this evaluation were correlated with the results of the semiquantitative analysis of GAD immunoreactivity performed by Mugnaini and Oertel. Specific labeling was only observed in neuronal cell bodies, whereas no labeling was found over neuropil, glial and endothelial cells. The highest labeling was found in the bulbus olfactorius (internal plexiform and granular layers) and in the caudal magnocellular nucleus of the hypothalamus. Strong labeling was observed in the Purkinje layer of the cerebellar cortex, the interpeduncular nucleus, the interstitial nucleus of Cajal, the nucleus of Darkschewitsch and the suprachiasmatic nucleus. Intermediate or low levels of GAD mRNA were present in various brain nuclei, where gamma-aminobutyric acid (GABA)-containing cell bodies had been observed with other techniques. Interestingly, a low level of GAD mRNA was found in the caudate-putamen and nucleus accumbens, where the vast majority of nerve cells is known to contain GAD immunoreactivity. Only a poor correlation was found between the present semiquantitative measurements of GAD mRNA content and previous analyses of the number of GAD-immunoreactive cell bodies. The present study demonstrates that there exists a differential regional expression of GAD mRNA. The comparison with cell counts performed by immunocytochemistry suggests that some brain areas, such as caudate-putamen and nucleus accumbens, contain a large number of GAD-immunoreactive cell bodies which express a low level of GAD mRNA. The opposite seems to be true for other nuclei, such as the globus pallidus, the zona reticulata of the substantia nigra and the inferior collicle, where few GAD-immunoreactive cell bodies contain high levels of GAD mRNA. In conclusion, the present study gives a low magnification map of GAD mRNA levels in the adult male rat brain. Marked biochemical heterogeneities may be present among GABA neuronal populations based on their expression of GAD mRNA. The comparison between the present in situ hybridization and previous immunocytochemical studies suggests that there may exist at least two populations of GABA neurons in the brain, having high and low levels respectively of both GAD mRNA and GAD enzyme.

Animals↗
PubMed1996

Diagnostic sensitivity of immunodominant epitopes of glutamic acid decarboxylase (GAD65) autoantibodies in childhood IDDM.

The prevalence and titre of epitope-specific autoantibodies to glutamic acid decarboxylase (GAD65) in 155 insulin-dependent diabetic (IDDM) and 9 GAD65 antibody (Ab)-positive healthy children were determined using four GAD65/67 chimaeric molecules which discriminate among the N-terminal (N), middle (M) and C-terminal (C) epitopes of GAD65. Radioligand binding assays for IgGAb used immunoprecipitation of in vitro translated 35S-GAD. We found autoantibodies to GAD65 in 116 of 155 (75%), to GAD67 in 19 of 155 (12%) (p < 0.0001) and to the GAD65-N-67 chimaera in 25 of 155 (16%) (p < 0.0001) IDDM sera. GAD67Ab were found almost exclusively (17 of 19, 89%) in GAD65Ab-positive sera and the levels of GAD67Ab correlated with those of GAD65Ab (r2 = 0.5913; p = 0.009). GAD65Ab directed to GAD65-M were found in 104 of 155 (67%), to GAD65-C in 104 of 155 (67%) and to GAD65-M + C in 116 of 155 (75%) of IDDM sera, and indicated reactivity to at least two distinct epitopes. Among the nine GAD65Ab-positive healthy children, two (22%) were also positive with GAD67, nine (100%) with GAD65-M + C, seven (78%) with GAD65-M, eight (89%) with GAD65-C and two (22%) with GAD65-N-67. Titres of GAD65Ab (p = 0.007), GAD65-C-Ab (p = 0.002) and GAD65-C + M-Ab (p = 0.003), but not of GAD65-M-Ab (p = 0.101) were significantly higher in IDDM than in healthy children. We conclude that GAD65Ab in IDDM and healthy children are directed to middle and C-terminal epitopes, and propose that levels of antibodies specifically directed to the carboxy-terminal end of GAD65 may distinguish IDDM from healthy children.

Adolescent↗
PubMed1999

An endogenous activator of renal glutamic acid decarboxylase effects of adenosine triphosphate, phosphate and chloride on the activity of this enzyme.

The renal glutamic acid decarboxylase (GAD) differs from the brain and pancreatic enzyme by its strong binding to membranes that is not influenced by detergents. After centrifugation of freshly prepared homogenate of the rat renal cortex, only 10-15% of GAD activity was found in supernatants and 15-30% in pellets. The majority of the GAD activity was lost. The bound GAD was found in the pellet. A thermolabile activator was present in the supernatant, which was not lost on dialysis. Approximately 55% of the total GAD activity was solubilized in homogenates stored for 24 h at 4 degrees C without detergent, whereas in homogenates stored with Triton X-100, the solubilized GAD increased to 80%. This solubilization was decreased by inhibitors of thioproteases such as leupeptin, antipain and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64). Solubilized GAD was applied to DEAE Toyopearl resin and the GAD activator was eluted with 35 mM Pi. GAD was eluted with 250 mM Pi. The effect of ATP on the activity of renal GAD was also different to its effect on brain GAD. ATP is a strong inhibitor of the brain enzyme at physiological concentrations. ATP (and Pi), together with chlorides (another brain GAD inhibitor), stabilize the renal GAD. However, renal GAD was inhibited by ATP in the presence of leupeptin in freshly prepared homogenates. Similarly, ATP inhibits solubilized GAD from homogenates stored without Triton X-100 for 24 h at 4 degrees C, but Pi retains its stabilizing effect in this preparation. A significant finding of the work presented here is the obligatory requirement of an endogenous activator for renal GAD activity. Whether this activator is an enzyme converting the inactive GAD to active enzyme (as hypothesized for brain GAD), or whether it is a protein affecting the activity of renal GAD by binding (as observed for GAD in some plants) remains to be established.

Adenosine Triphosphate↗
PubMed2000

Glutamic acid decarboxylase-positive neuronal cell bodies and terminals in the human cerebellar cortex.

The distribution of gamma-aminobutyric acid (GABA) in the human cerebellar cortex was studied using immunohistochemistry for glutamic acid decarboxylase (GAD), the enzyme that catalyses GABA synthesis. Observations by light microscopy revealed, in all layers of the cerebellar cortex, strong, punctate positivity for GAD, related to putative GABAergic nerve terminals, as well as a diffuse cytoplasmic immunoreactivity within neuronal cell bodies. GAD-positive nerve terminals were found in close relationship with the walls of the cerebellar cortex microvessels. Observations by electron microscopy revealed positive nerve terminals in contact with the astrocyte perivascular sheath of capillaries. GAD immunoreactivity was also detected within astroglial perivascular endfeet and endothelial cells. The findings provide further insights into the GABAergic synapses of the circuitry of the human cerebellar cortex. The detection of 'vascular' GAD immunoreactivities suggests that GABAergic mechanisms may regulate cerebellar microvessel function.

Aged↗
PubMed1992

Decreased tissue reaction to bioprosthetic heart valve material after L-glutamic acid treatment. A morphological study.

Degenerative alterations of two different glutaraldehyde (GA)-fixed bioprosthetic heart valve materials were investigated in subcutaneous rat implants: Bovine pericardium, prepared according to clinically used bioprosthetic heart valve material (BHV) was compared to alternatively preserved pericardium (APHV), which was fixed in GA and treated with L-glutamic acid. Following 63 days of subcutaneous implantation, calcification of APHV implants was significantly lower as compared to BHV implants (13 +/- 6 versus 158 +/- 18 micrograms Ca/mg dry weight tissue; p less than 0.05). In BHV implants ultrastructural investigations showed nucleation of plate-shaped hydroxyapatite crystals at the surface of collagen fibrils and in remnants of connective tissue cells; no signs of calcification could be detected in APHV implants. The time-course of the inflammatory reaction was determined by quantification of immunohistochemical stained mononuclear host-cells invading the implants. In both preparation groups inflammatory reaction reached maximum 42 days after implantation. However, infiltration rate of inflammatory cells was markedly decreased in APHVs as compared to BHVs (p less than 0.05).

Animals↗
PubMed1988

Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase.

Immunoaffinity columns are prepared from the monoclonal antibody (MAb) GAD-1. These columns are used to enrich glutamic acid decarboxylase (GAD) from the cytosolic fraction of rat brain homogenates and from Triton X-100 extracts of the brain membrane fraction. In each case enzyme activity is enriched over 400-fold. The immunopurified fractions were analyzed by SDS-PAGE. Fractions purified from the cytosol consisted of a quantitatively major band of 59 kDa, and one band of 63 kDa, as well as a group centered around 55 kDa. Fractions purified from membranes consisted primarily of the 59 and 63 kDa components; only traces of the lower-molecular-weight components were present. The entire set of proteins purified on GAD-1 immunoaffinity columns is strongly recognized by 2 widely used antisera to GAD, those described in Saito et al. (1974) and Oertel et al. (1981). The 59 kDa protein from the cytosolic fraction was purified to homogeneity by preparative SDS-PAGE; a partial amino acid sequence of this protein was obtained. The 59 kDa protein has a high degree of sequence homology with the deduced amino acid sequence of the protein that was coded for by a cDNA for feline GAD (Kaufman et al., 1986; Kobayashi et al., 1987). Thus, these proteins are either products of a single gene that diverged during the evolution of rat and cat from a common ancestor, or are members of a closely related set of genes found in both species. The MAb GAD-6 recognizes the 59 kDa band and the group of bands centered around 55 kDa on Western blots. Therefore, these proteins are immunochemically related. GAD-6 does not recognize the 63 kDa band. In Western blots of unfractionated homogenates of the whole brain, the only band recognized by GAD-6 is a 59 kDa band.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1998

Hemostatic capability of rapidly curable glues from gelatin, poly(L-glutamic acid), and carbodiimide.

The hemostatic capability of rapidly curable glues composed of gelatin and poly(L-glutamic acid) (PLGA) was compared with that of the conventional fibrin glue. The hydrogels produced from mixed gelatin and PLGA aqueous solution within several seconds by addition of water-soluble carbodiimide (WSC) was applied to the dog spleen injured by needle pricking. The WSC-catalyzed gelatin-PLGA glues exhibited higher hemostatic capability than the fibrin glue. The total amount of bleeding from the injured spleen until hemostasis when the gelatin-PLGA hydrogel glues were applied was significantly smaller than that of the fibrin glue application. The gelatin-PLGA glue application enhanced the success rate of complete hemostasis to a significantly greater extent than the fibrin glue, while the frequency of glue applications until achieving complete hemostasis decreased. The gelatin PLGA hydrogels strongly adhered to the surface of dog spleen, whereas the fibrin hydrogel was easily detached from the spleen surface. It was concluded that this strong adhesion mechanically suppressed the bleeding, leading to enhanced hemostasis by the rapidly curable gelatin-PLGA glues.

Animals↗
PubMed2003

Antifreeze activities of poly(gamma-glutamic acid) produced by Bacillus licheniformis.

Various enantiomeric isomers, metals salts and molecular sizes of poly(gamma-glutamic acid), gamma-PGA, produced by Bacillus licheniformis CCRC 12826, were prepared and their antifreeze activities were studied by differential scanning calorimetry. The antifreeze activity of gamma-PGA increased as its molecular weight decreased but was indifferent to its D/L-glutamate composition. The antifreeze activity was cation dependent decreasing in the order Mg2+ >> Ca2+ approximately Na+ >> K+ which follows that of inorganic chlorides in that high ionic charge leads to high antifreeze activity. The mechanism by which the cryoprotective effects of gamma-PGA can be explained is still yet to be determined.

Antifreeze Proteins↗
PubMed2006

Synthesis of macrocyclic analogues of the neuroprotective agent glycyl-L-prolyl-L-glutamic acid (GPE).

The syntheses of seven macrocyclic analogues of the neuroprotective tripeptide glycyl-L-prolyl-L-glutamic acid (GPE) are described. Macrocycles 6 and 7 mimic the cis conformer of GPE whereas macrocycles 2-5, 8, and 9 mimic the trans conformer of GPE. The macrocyclic peptides of well-defined geometry were prepared via Grubbs ring closing metathesis of an appropriate diene precursor. In turn each of the diene precursors were prepared from the readily available allyl-substituted amino acid building blocks 12, 13, 14, 27, 36 and 51.

Macrocyclic Compounds↗
PubMed2000

Unfolding of poly-L-glutamic acid by microbubbling of supercritical carbon dioxide.

The conformational changes in alpha-helical poly-L-glutamic acid caused by microbubbling supercritical CO2 were investigated with circular dichroism spectra. After microbubbling using a micropore filter at 35 and 30 MPa for 30 min, alpha-helix content decreased to 37%, while without the filter it was 68%. The alpha-helix structure was significantly decomposed by a high density of CO2. No important changes were observed in heating, autoclaving, or pH-lowering.

Carbon Dioxide↗
PubMed1994

Drosophila GABAergic systems. II. Mutational analysis of chromosomal segment 64AB, a region containing the glutamic acid decarboxylase gene.

The Drosophila melanogaster Gad gene maps to region 64A3-5 of chromosome 3L and encodes glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Because this neurotransmitter has been implicated in developmental functions, we have begun to study the role of GABA synthesis during Drosophila embryogenesis. We show that Gad mRNA is expressed in a widespread pattern within the embryonic nervous system. Similarly, GAD-immunoreactive protein is present during embryogenesis. These results prompted us to screen for embryonic lethal mutations that affect GAD activity. The chromosomal region to which Gad maps, however, has not been subjected to an extensive mutational analysis, even though it contains several genes encoding important neurobiological, developmental, or cellular functions. Therefore, we have initially generated both chromosomal rearrangements and point mutations that map to the Drosophila 64AB interval. Altogether, a total of 33 rearrangements and putative point mutations were identified within region 64A3-5 to 64B12. Genetic complementation analysis suggests that this cytogenetic interval contains a minimum of 19 essential genes. Within our collection of lethal mutations are several chromosomal rearrangements, two of which are in the vicinity of the Gad locus. One of these rearrangements, Df(3L)C175, is a small deletion that removes the Gad locus and at least two essential genes; the second, T(2;3)F10, is a reciprocal translocation involving the second and third chromosomes with a break within region 64A3-5. Both of these rearrangements are associated with embryonic lethality and decreased GAD enzymatic activity.

Animals↗
PubMed1998

N-benzoyl, L-glutamic acid as a suitable internal standard for the analysis of trans,trans-muconic acid in human urine by liquid chromatography.

Urinary trans,trans-muconic acid is a sensitive biomarker for low level benzene exposure. The method described by Ducos et al. (Int Arch Occup Environ Health 1990; 62:529-34) is commonly used for its determination. In this study, we demonstrate that N-benzoyl, L-glutamic acid added to urine samples is a suitable internal standard to control trans,trans-muconic acid recovery after solid phase extraction of urine and to compensate for variations which might occur during high-performance liquid chromatography analysis.

Benzene↗
PubMed1995

In vitro stimulation with glutamic acid decarboxylase (GAD65) leads to an oligoclonal response of peripheral T-cells in an IDDM patient.

The enzyme glutamic acid decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). To study T-cell reactivity towards GAD, peripheral blood leucocytes from seven patients with IDDM and five control subjects were stimulated in vitro with recombinant GAD. All diabetics studied were heterozygous for diabetes-associated HLA alleles, i.e. HLA-DRB1*03,*04-DQB1 *0302,*0201. A single IDDM subject (no. GAD65.05) revealed a strong response against GAD65. After stimulation, his T-cell receptor beta (TCRBV) usage was found to be oligoclonal. The sequence analysis of the putative peptide binding region of the T-cell receptor (CDR3 region) of 37 GAD-reactive T-cell clones revealed no common CDR3 motif. The stimulation of GAD-reactive T-cells could be inhibited with anti-class II monoclonal antibodies, indicating a class II restricted T-cell response. In addition, GAD65-responsive T-cells revealed a Th1 cytokine response pattern. The author's data suggest that GAD-reactive T-cells of Th1 phenotype can be obtained after in vitro stimulation of peripheral blood leucocytes from an HLA-DRB1*03/*04 heterozygous IDDM patient. The lack of a common CDR3 motif suggests the absence of an immunodominant T-cell epitope in that patient, or may indicate receptor repertoire spreading of peripheral T-lymphocytes.

Amino Acid Sequence↗