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PubMed1999

Competitive inhibition of a glutamate carboxypeptidase by phosphonamidothionate derivatives of glutamic acid.

Several N-thiophosphonyl-glutamates were found to be potent competitive inhibitors of a zinc-dependent glutamyl hydrolase, carboxypeptidase G (CPG). Weak inhibition exhibited by an analogous N-phosphonyl-glutamate suggests that the enhanced potency of the phosphonamidothioates is due to the presence of their sulfur ligand and its favorable interactions with active site features, presumably zinc(II).

Carboxypeptidases↗

PubMed2003

Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs.

Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.

Amino Acid Motifs↗

PubMed2005

Medium optimization by response surface methodology for poly-gamma-glutamic acid production using dairy manure as the basis of a solid substrate.

Dairy manure, supplemented with agro-industrial materials, was used as the solid substrate for high yield of poly-gamma-glutamic acid (gamma-PGA) by Bacillus subtilis CCTCC202048. The solid-state fermentation medium was optimized by response surface methodology. In the first optimization step, a Plackett-Burman design was used to evaluate the influence of related factors. Wheat bran, soybean cake and glutamic acid were found to be more compatible supplement with dairy manure and positively influenced on gamma-PGA production. In the second step, the concentrations of the three supplemental nutrients above were further optimized using a Box-Behnken design. The average gamma-PGA yield (4.70%) in triplicate under optimal conditions was obtained on the laboratory scale, whereas it was 3.58% at compost experiment. These would lay a foundation for lessening the pollution of dairy manure, increasing fertilizer efficiency and exploring a late-model organic fertilizer that retains water and nutrients.

Animals↗

PubMed1993

Expression of the genes coding for glutamic acid decarboxylase in pluripotent cell lines.

The expression of glutamic acid decarboxylase (GAD) is a basic characteristic of a wide array of inhibitory neurons the use gamma-aminobutyric acid as a neurotransmitter. Clonal cell models will be essential for investigating the mechanisms which are responsible for the selective expression of GAD. P19 embryonal carcinoma cells are an important model for the analysis of neuronal gene expression. Depending on culture conditions, undifferentiated cells can be induced to form cells as widely divergent as cardiac muscle-like cells and neuron-like and glial-like cells. P19 cells are amendable to a number of powerful genetic manipulations including transformation with foreign DNA and selection of mutants. In this study we used nuclease protection assays and Northern blot analysis to determine if P19 cells express the GAD1 and GAD2 genes. The results show that uninduced P19 cells express these genes at very low but easily detectable levels. When the cells are induced to differentiate along the neuronal pathway with retinoic acid, the levels of transcripts for both GAD genes rise dramatically. At least some RNA transcripts of both genes from induced cells comigrate with the corresponding mRNA from the brain and thus probably represent processed mRNA. The expression of GAD genes in undifferentiated cultures of embryonal stem (ES) cells was also investigated. These cultures express levels of GAD1 transcripts that are higher than uninduced P19 cells. In contrast, expression of the GAD2 gene was barely detectable. These results indicate that P19 EC cells and ES cells will be useful for the investigation of the mechanisms that regulate the expression of the GAD1 and GAD2 genes.

Animals↗

PubMed1995

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues.

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al3+, Ca2+, Fe3+, and Yb3+ and the borderline metal ion Cu2+, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the range pH 5.5-8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized Mn+-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe3+-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe3+-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metal-ion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surface-exposed aspartic and glutamic acid residues on the protein with the immobilized Mn+-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu2+-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number of clustering of the surface-accessible aspartic and glutamic residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.

Animals↗

PubMed1986

Monoclonal antibodies to glutamic acid decarboxylase.

Five monoclonal antibodies that recognize chicken brain glutamic acid decarboxylase (GAD) have been selected and designated GAD-1 to -5. GAD-1 to -5 were selected on the basis of their ability to immunoprecipitate active GAD from crude brain extracts. GAD-1 recognizes an epitope that is conserved in many vertebrates; the epitope recognized by GAD-5 is restricted to the chicken. Radioimmunoassays with GAD-1 indicate that GAD is highly enriched in brain relative to other tissues. GAD was localized immunocytochemically with GAD-1 and GAD-2 in rat cerebellum, spinal cord, and retina. The staining pattern is in agreement with that obtained previously with polyclonal antisera to GAD. GAD from the chicken brain was purified by chromatography on an immunoaffinity column made of GAD-1. NaDodSO4/PAGE analysis of the immunoaffinity-purified GAD fractions shows a major band of 59 kDa and minor bands at 63 and 54 kDa.

Animals↗

PubMed1977

The gamma-carboxy glutamic acid content of human and bovine prothrombin following warfarin treatment.

A form of prothrombin induced by Warfarin therapy, has been isolated which is adsorbed onto insoluble barium salts, but has a reduced biological activity. This protein contains, on average, seven out of a possible ten gamma-carboxy glutamic acid residues. A second form of prothrombin is also described, which is not adsorbed into barium slats, and has less than 1% the activity of the normal protein, contains only four gamma-carboxy glutamic acid residues. The significance of these results is discussed.

Amino Acid Sequence↗

PubMed1996

Diabetic sera react with the glutamic acid decarboxylase molecule in a dimeric-oligomeric form.

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). This was initially identified as a 64-65 kD molecule according to migration in gels after immunoprecipitation from pancreatic islets. We studied the antigenicity of two different radiolabelled preparations of GAD, derived either by affinity purification from porcine brain and known to contain GAD 65 and GAD 67, or by expression from a cDNA for human GAD 65 by rabbit reticulocyte lysate (RRL). Radiolabelled immunoprecipitated pellets from the reaction of potent antisera to GAD from patients with IDDM were examined by autoradiography after SDS-PAGE under reducing or non-reducing conditions. Also, preparations of porcine brain GAD were "depleted' of GAD by exposure to antisera, and then similarly re-examined. Autoradiography of radiolabelled GAD either affinity purified from porcine brain, or expressed by RRL, showed that the immunoprecipitated protein migrated under non-reducing conditions according to a M(r) of approximately 110-130 kD, corresponding to dimeric forms of monomeric GAD of approximately 55-65 kD. Depletion by immunoprecipitation of this minor higher M(r) component from preparations of GAD left, in the supernatant, an abundance of GAD of M(r) 64-65 kD corresponding to monomer that was completely non-reactive with potent IDDM sera. We conclude that IDDM sera react with the GAD molecule in a dimeric (or oligomeric) form. Our findings have general connotations for self-tolerance and autoimmunity.

Animals↗

PubMed2003

Improved in planta expression of the human islet autoantigen glutamic acid decarboxylase (GAD65).

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH2-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67(1-87)/GAD65(88-585) molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.

Autoantigens↗

PubMed1998

Antibodies to glutamic acid decarboxylase in young Chinese diabetic patients.

Antibodies to glutamic acid decarboxylase (GAD) are a useful autoimmune marker for type 1 diabetes mellitus in Caucasians. We examined antibodies to GAD and their relationships with clinical features and pancreatic beta cell function in 140 young Chinese diabetic patients. Over an 18-month period beginning in 1995, 140 young Chinese diabetic subjects with age of onset of disease < or = 35 years and age < 40 years were recruited consecutively, irrespective of their modes of presentation. Clinical features, antibodies to GAD and pancreatic beta cell function (using a glucagon stimulation test) were examined. Increased levels of antibodies to GAD (> 18 units) were detected in 12.1% (n = 17) of these subjects. Forty-three (31%) patients had a classical type 1 presentation and 65 (46%) patients were insulin-deficient based on post-glucagon plasma C-peptide levels. Patients who were insulin-deficient and had a type 1 presentation had the highest prevalence of antibodies to GAD (29.0%) compared with patients who had a type 2 presentation and were non-insulin deficient (6.4%, P = 0.003). Patients who had antibodies to GAD had lower body mass index and waist-hip ratio, earlier onset of disease, lower blood pressure, plasma triglyceride and C-peptide, and higher concentrations of plasma high-density lipoprotein cholesterol and glycated haemoglobin, and were more likely to require drug treatment, compared with those without antibodies to GAD. In conclusion, there was a low prevalence of antibodies to GAD in Chinese young diabetic patients although such antibodies remained a relatively specific marker for insulin deficiency and acute presentation. Causes other than autoimmunity should be sought to explain the high prevalence of insulin deficiency in these young Chinese patients.

Antibodies↗

PubMed1996

Clinical evaluation of non-insulin-dependent diabetes mellitus patients with autoantibodies to glutamic acid decarboxylase.

We evaluated the frequency of antibodies to glutamic acid decarboxylase (GAD-Ab) in Japanese patients diagnosed initially as having non-insulin-dependent diabetes mellitus (NIDDM) and investigated a possible link between the presence of GAD-Ab and development of the insulin-dependent (ID) state. The population sample consisted of 583 Japanese NIDDM patients (age at onset > 30 years) who were initially non-ketotic and did not require insulin treatment during at least 6 months of observation. GAD-Ab were measured using radioimmunoassay. The clinical characteristics of GAD-Ab+ patients were carefully examined at four-year intervals from the onset of diabetes. We also examined the ID state by measuring the level of postprandial serum C-peptide and i.v. glucagon-stimulated serum C-peptide. The overall prevalence of GAD-Ab in Japanese NIDDM patients was 3.8%. The frequency of GAD-Ab+ did not significantly decrease with a long history of diabetes. GAD-Ab+ patients had a lower body mass index, compared with GAD-Ab- (20.8 +/- 2.9 vs 23.0 +/- 3.7, P < 0.005), lower postprandial C-peptide levels (0.7 +/- 0.6 vs 1.4 +/- 1.2, P < 0.01), and an early commencement of insulin therapy (3.6 +/- 4.7 vs 8.3 +/- 6.6, P < 0.01). GAD-Ab+ patients who had already developed the ID state had characteristically higher titers of GAD-Ab (421.4 +/- 359.1) and a higher frequency of islet cell antibodies (ICAs) (77.8%), compared with GAD-Ab+ NID patients (titer: 60.2 +/- 86.9, P < 0.005, 23.1%, P < 0.05, respectively). GAD-Ab+ ICAs+ patients showed higher frequencies of ID state at any diabetic duration compared with GAD- ICAs-, while GAD-Ab+ ICAs- patients did not differ in the frequency of the ID state from GAD- ICAs-. Our results suggest that the presence of both GAD-Ab and ICAs represents a high risk for IDDM in GAD-Ab+ NIDDM patients.

Adult↗

PubMedDate not supplied

[Effect of glutamic acid on Saccharomyces cerevisiae cell permeability during rehydration after dehydration].

Losses of potassium ions, nucleotides and protein substances were studied upon rehydration of Saccharomyces cerevisiae cells grown on a defined nutrient medium with ethanol and glutamic acid (0.075%) and subjected to dehydration. Considerable amounts of K+ (75--82% of the total), nucleotides and protein substances were found to be released into distilled water upon rehydration of the dehydrated yeast cells. If a higher percentage of yeast cells survived, these lost less nucleotides and nitrogen-containing substances. Glutamic acid being added to the growth medium did not decrease specifically the permeability of the dehydrated yeast cells upon rehydration.

Adsorption↗

PubMedDate not supplied

Novel functional biodegradable polymer: synthesis and anticoagulant activity of poly(gamma-glutamic acid)sulfonate (gamma-PGA-sulfonate).

gamma-Poly(glutamic acid) (gamma-PGA), which is produced by Bacillus subtilis, was sulfonated using 2-aminoethane-1-sulfonic acid (taurine) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (WSC) to give sulfonated gamma-PGA (gamma-PGA-sulfonate). From (1)H NMR spectroscopy and IR spectroscopy, it was confirmed that taurine was introduced to the side chain of gamma-PGA via an amide linkage. By altering the synthetic conditions, it was possible to control the content of sulfonate in gamma-PGA-sulfonate. Anticoagulant activity was investigated in order to evaluate the biological activity of gamma-PGA-sulfonate by the Lee-White test. The clotting time was prolonged when the concentration of gamma-PGA-sulfonate on the degree of sulfonation was increased. It becomes clear that gamma-PGA-sulfonate is potentially useful for various medical applications, such as drug delivery, tissue engineering, and medical materials.

Anticoagulants↗

PubMed2002

Direct observation of the multistep helix formation of poly-L-glutamic acids.

The helix formation dynamics of poly-L-glutamic acids (PGAs) were observed by the microsecond-resolved Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. The helix formation of 34-residue PGA from random coil at pH (or pD for FTIR) 8.0 was initiated by a pH jump to 4.9 using the rapid solution mixer whose mixing dead time is 50 micros. The amide I' line in the time-resolved FTIR spectra exhibited the fast (<100 micros) increase of the total helical content. The time-resolved CD spectra of the same process also showed the fast (<150 micros) formation of short helical segments (5 +/- 1 residues), which was followed by the slower (<1 ms) elongation of the short helices to longer helices (>10 residues). Similar dynamics were observed for the same pH jump of approximately 190-residue PGA, although there were additional steps that made the helix formation of approximately 190-residue PGA more complex. The observed multistep helix formation is likely caused by the strong hydrogen-bonding interactions between the protonated side chains of PGAs.

Circular Dichroism↗

PubMed2002

Participation of the cholinergic system in the excessive grooming behavior induced by neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI).

The neuropeptide (N) glutamic acid (E) isoleucine (I) amide (NEI) injected into the ventral tegmental area (VTA) or intraventricularly (icv) induces excessive grooming behavior (EGB) and motor activity (MA). Here, we studied whether the cholinergic system is involved in the NEI-induced behavior. The present results demonstrate that atropine, a general muscarinic antagonist, injected icv previous to NEI, suppresses the behavior provoked by icv injections of the peptide, whereas the prior icv injection of dyhidro-beta-erythroidine, a general nicotinic antagonist, did not affect the EGB and MA induced by the peptide. From the experimental evidence, it is suggested that NEI may act specifically on a cholinergic afferent to dopaminergic cells. Also, the results appear to indicate that a neural target, different from the dopamine system, may be activated by the peptide to elicit behavioral changes, such as EGB.

Animals↗

PubMedDate not supplied

[Experience in using glutamic acid electrophoresis in the early treatment of craniocerebral injuries].

Endonasal electrophoresis with alpha-glutamic acid was used in 76 patients with severe craniocerebral injury. The result was good in 24 (38%) and satisfactory in 40 (55%) patients. Thus, a positive result of treatment was obtained in 88% of cases, which was confirmed by electrophysiological and neuropsychological studies. The method produced the greatest effect on the patients' psychic status: 74% of all disorders of higher psychic functions, mainly memory, were restored on the average by the end of treatment. Good tolerance of electrophoresis and its efficacy in the early period after severe injury, i. e. in the first month, were established. Contraindications for treatment were defined more precisely. Since the method is simple and does not call for special conditions, it may be widely used in any medical establishment equipped with galvanization apparatuses.

Administration, Intranasal↗

PubMed1990

Regional hemodynamic effects produced following application of L-glutamic acid to the ventral medullary surface of the cat.

The neuronal stimulant L-glutamic acid (GA) was applied to the ventral medullary surface and diastolic arterial blood pressure (DBP), heart rate (HR), renal and femoral arterial blood flow were measured in the chloralose-anesthetized cat. GA (1.0 M) increased DAP by 27 +/- 6.6 mmHg (P less than 0.05, n = 6), but heart rate was not affected (P greater than 0.05). Renal arterial resistance (RR) increased by 15 +/- 5.8% (P less than 0.05) and femoral resistance (FR) increased by 46 +/- 10% (P less than 0.05). The difference in the change in RR and FR was statistically significant (P less than 0.05). These effects were dose-dependent and could be blocked by prior intravenous administration of the alpha-blocker prazosin. These data indicate that neurons in the intermediate area of the ventromedullary surface may have a greater control over femoral flow than renal flow.

Adrenergic Fibers↗

PubMedDate not supplied

Conformational study in water by NMR and molecular modeling of cyclic glutamic acid analogues as probes of vitamin K dependent carboxylase.

The conformational analysis of four glutamic acid analogues containing a cyclopentyl or cyclohexyl ring, substituted in position 1 by a BOC protected amino group and a methyl ester group and in position 3 by a free carboxylate group, has been carried out in an aqueous environment, by 1H and 13C NMR spectroscopy, and molecular dynamics (MD). Their structural properties were under investigation for a structure-activity relationship analysis to determine the preferred conformation in the carboxylase active site. For each compounds, resulting conformations from NMR and MD data were analyzed and classified according to the dihedral angles chi 1 and chi 2, the distances and the spatial distribution involving charged or substituted C- and N-terminal groups. A reduced number of conformational families were found to be in qualitative agreement with NMR and MD data. A comparison between these different classes of the active and nonactive derivatives was achieved.

Animals↗