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A tetrapeptide, residues 6 to 9 in normal prothrombin, was isolated from the NH(2)-terminal, Ca(2+)-binding part of normal prothrombin. The electrophoretic mobility of the peptide was too high to be explained entirely by its amino-acid composition. According to (1)H nuclear magnetic resonance spectroscopy and mass spectrometry, the peptide contained two residues of modified glutamic acid, gamma-carboxyglutamic acid (3-amino-1,1,3-propanetricarboxylic acid), a hitherto unidentified amino acid. This amino acid gives normal prothrombin the Ca(2+)-binding ability that is necessary for its activation. Observations indicate that abnormal prothrombin, induced by the vitamin K antagonist, dicoumarol, lacks these modified glutamic acid residues and that this is the reason why abnormal prothrombin does not bind Ca(2+) and is nonfunctioning in blood coagulation.
Glutamic acid decarboxylase (GAD) activity was measured in the oviduct of normal rats in diestrous and in rats ovariectomized (OVX) seven days before. OVX induced a significant decrease of GAD activity in the Fallopian tube. This effect was completely reversed by coadministration of estradiol benzoate + progesterone (E + P). Simultaneous injection of atropine, but not of alpha-methyl-para-tyrosine or labetalol, completely prevented the activation of GAD induced by ovarian sterois. Moreover, prostigmin significantly potentiated the action of E + P on GAD activity in the rat oviduct. These data clearly suggest the participation of acetylcholine in the mechanisms whereby ovarian steroids regulate GAD activity in the rat Fallopian tube.
Immunohistochemical experiments were performed using glutamic acid decarboxylase (GAD) to identify gamma-aminobutyric acid (GABA)ergic neurons in the vestibular nuclei (VN). VN neurons projecting to the sensory trigeminal complex (STC) or to the C1-C2 segments of the spinal cord were identified by injection of wheat germ agglutinin-apo-horseradish peroxidase coupled to colloidal gold (gold-HRP), a retrogradely transported tracer, in these structures. The experiments combining injection of gold-HRP in spinal cord and GAD immunohistochemistry revealed the existence in the medial, inferior and lateral VN of GAD immunoreactive neurons projecting to the spinal C1-C2 level. Experiments combining injection of gold-HRP in the STC and GAD immunohistochemistry demonstrated that, at least, 30-50% of the vestibulo-trigeminal neurons also contained GAD. Injections of two different retrograde tracers (gold-HRP and Biotinylated dextran amine) in the STC and the spinal cord demonstrated that some VN neurons project by axon collaterals to both structures. Because of the GABAergic spinal and STC vestibular projections we assume that these VN neurons with collateral projection are GABAergic. Therefore primary afferents from the face, neck or hindlimb could be modulated by inhibitory influences from GABAergic vestibular neurons.
Glutamic acid decarboxylase antibodies (GAD65Ab) are common in new onset Caucasian insulin-dependent diabetic (IDDM) patients but it is unclear if this marker is also prevalent in patients of other ethnic backgrounds. We determined antibodies against human recombinant GAD in Japanese diabetic patients using a radioimmunoassay with competition between in vitro translated 35S-GAD65 and non-labelled recombinant human GAD65 (rhGAD65). GAD67 antibodies (GAD67Ab) were similarly analyzed but without antigen competition. In 73 Japanese diabetic patients, GAD65Ab were found in 11/16 (69%) of patients with short-duration (less than 5 yrs) IDDM, 6/23 (26%) with long-duration (5 or more yrs) IDDM and 10/20 (50%) with slowly progressive diabetes. High GAD65Ab levels were associated with concomitant autoimmune diseases (p = 0.021). GAD67Ab were found in 4/16 (25%) of patients with short-duration IDDM, 3/23 (13%) with long-duration IDDM and 2/20 (10%) with slowly progressive diabetes. In 14 non-insulin dependent diabetic (NIDDM) patients, GAD65Ab and GAD67Ab were not found (0/14) and 1/50 (2%) healthy controls were positive in either assay. Among the GAD67Ab-positive samples, 8/9 (88%) were also high level GAD65Ab positive, 7/9 (77%) were displaced by an excess of rhGAD65 and the antibody levels correlated (r2 = 0.573; p = 0.003). Our data are consistent with a strong association of GAD65Ab also in Japanese IDDM, and suggest that, when present, GAD67Ab are frequently directed to epitope(s) common to GAD65 and GAD67.
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Glycine and glutamic acid were found to exert an effect on the processes of abomasal emptying. Following glycine administration, a significant inhibition of the evacuation processes occurred with its subsequent activation.
In continuation of previous studies, the intraarterial fusion of L-glutamic acid for 24 hr was found to oppose the decrease in acetylcholinesterase and butyrylcholinesterase in the superior cervical ganglion of the cat that otherwise occurs 48 hr after preganglionic denervation. The combination of glutamic acid and gamma-aminobutyric acid, in concentrations that were inactive individually, likewise produced the same neurotrophic effect. Inactive in this respect were glycine plus L-glutamine, pyroglutamic acid, gamma-aminobutyric acid, and L-aspartic acid. The possible mechanisms and implications of these findings are discussed.
Poly-gamma-glutamic acid (gamma-PGA) is a very promising biodegradable polymer that is produced by Bacillus subtilis. Gamma-PGA is water-soluble, anionic, biodegradable, and edible. This paper reviews the production of a strain of gamma-PGA and recent developments with respect to applications in terms of Ca absorption, moisturizing properties, gamma-PGA conjugation, super absorbent polymer, and so on. Our recent research shows that gamma-PGA can be used as an immune-stimulating and anti-tumor agent, especially at high molecular weight.
1. Pyramidal tract interneurones, defined as neurones which are activated synaptically as a result of pyramidal tract stimulation, have been identified in the rat cerebral cortex. The number of evoked spikes depended upon stimulus strength, and stimulation in a specific thalamic nucleus produced a burst of activity lasting for up to 1 sec.2. These cells are readily excited by a brief (50 msec) pulse of glutamate applied by micro-iontophoresis. Other, unidentified cells are not so responsive.3. Synaptically evoked spikes resulting from pyramidal tract stimulation can be blocked by the iontophoretic and I.P. administration of substances shown to antagonize glutamate excitation of cells.4. The results support suggestions that glutamic acid is a neurotransmitter in the cerebral cortex. The evidence presented further indicates that glutamic acid could be the transmitter released by the pyramidal tract.
The development of the specific binding sites for L-[3H]glutamic acid (KD = 370 nM) and for [3H]kainic acid (KD = 39 nM) was studied in the rat cerebellum. Specific binding at both sites remains low during the first week after birth but increases markedly during the second and third weeks after birth, when glutamatergic parallel fiber synaptogenesis occurs. The development of the kainate site lags behind that of the glutamate site, indicating their autonomy.
Human apolipoprotein (apo) E contains an amino- and a carboxyl-terminal domain, which are connected by a hinge region (approximately residues 165 to 215). The interaction of the two domains has been suggested to be responsible for the apoE4-binding preference for very low density lipoproteins (VLDL). In the absence of this interaction in apoE3, the preference is for high density lipoproteins (HDL). To exclude the possibility that the interaction of apoE with other apolipoproteins on the native particles may contribute to the isoform-specific preferences, VLDL-like emulsion particles were incubated with apoE, and the lipid-bound apoE was separated from free apoE on a Superose 6 column. The apoE4 bound more effectively to these particles than did apoE3, indicating that the apoE4 preference for VLDL is due not to interactions with other apolipoproteins but to an intrinsic property of apoE4, likely related to domain interaction. Previously, arginine 61 was shown to be critical for the isoform preferences, suggesting that it interacted with an acidic residue(s) in the carboxyl terminus. Substitution of arginine 61 with lysine did not alter the preference of apoE4 for VLDL, demonstrating that a positive charge rather than a specific requirement for arginine is critical for domain interaction. To identify the acidic residue(s) in the carboxyl terminus interacting with arginine 61, the six acidic residues (244, 245, 255, 266, 270, and 271) in a region known to be important for both lipoprotein association and isoform-specific preferences were substituted individually with alanine in apoE4. Only substitution of glutamic acid 255 altered the preference of apoE4 from VLDL to HDL, indicating that this was the sole residue in the carboxyl terminus that interacts with arginine 61. The participation of the hinge region in domain interaction was examined with internal deletion mutants. Deletion of the residues 186-202 or 186-223, representing major portions of the hinge region, had no effect on the apoE4 preference for VLDL. This suggests that the hinge region may act as a spacer that connects the two domains. Further deletion into the carboxyl-terminal domain (to residue 244) results in a loss of apoE4 VLDL binding. These studies establish that interaction of arginine 61 and glutamic acid 255 mediates apoE4 domain interaction.
Two mutant Chinese hamster lung fibroblast lines have been isolated that are resistant to the toxic proline analog L-azetidine-2-carboxylic acid. The line designated AZCA-1 has 30-fold elevated activity of pyrroline-5-carboxylate synthase and a large increase in the rate of proline production and release compared to controls. Pyrroline-5-carboxylate synthase activity is not elevated in the resistant line designated AZCA-4, but the enzyme is less sensitive to inhibition by ornithine and proline than control enzyme. Intracellular proline is elevated in AZCA-4 cells, with no change in the rate of release of proline synthesized from glutamate. Resistance to azetidine carboxylic acid in both mutant lines is attributed to the expanded intracellular proline pool that results from alterations in pyrroline-5-carboxylate synthase. These results indicate that intracellular proline levels are determined at least in part by the regulated activity of pyrroline-5-carboxylate synthase.
The syntheses of (RS)-alpha-amino-3-hydroxy-5-tert-butyl-4-isoxazolepropionic acid (9, ATPA), (alpha-RS, beta-RS)-alpha-amino-beta-methyl-3-hydroxy-5-isoxazolepropionic acid (8), (RS)-alpha-amino-3-hydroxy-5-isoxazolebutyric acid (15a), and (RS)-alpha-amino-3-hydroxy-5-isoxazolevaleric acid (15b) are described. The compounds were tested in vitro together with (RS)-alpha-amino-3-hydroxy-5-(bromomethyl)-4-isoxazolepropionic acid (ABPA) as inhibitors of the binding of radioactive-labeled (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to rat brain synaptic membranes. These data were compared with the earlier reported effects of the compounds on single neurons in the feline spinal cord obtained by microelectrophoretic techniques. The three compounds AMPA, ATPA, and ABPA are agonists at the class of receptors assumed to represent a subtype of physiological (S)-glutamic acid (Glu) receptors. Inhibition of [3H]AMPA binding by ATPA was 1 order of magnitude weaker than that of AMPA, in agreement with the relative potency of these compounds in vivo. ABPA proved to be equipotent with AMPA both as an inhibitor of AMPA binding and as a neuronal excitant. The compounds 8, 15a, and 15b have no effect as inhibitors of AMPA binding, in agreement with in vivo studies that have shown that 8 does not affect the firing of central neurons whereas 15a and 15b are antagonists at NMDA receptors, a subpopulation of excitatory receptors not affected by AMPA. Molecular mechanical calculations on AMPA, ATPA, and ABPA using the program MM2 showed that conformations of AMPA, ABPA, and especially ATPA by rotation of the amino acid side chain have energy barriers. A possible receptor-active conformation is suggested.
Antibodies to glutamic acid decarboxylase-65 (GAD65) are present in a number of autoimmune disorders, such as insulin-dependent (type 1) diabetes mellitus (IDDM), stiff man syndrome, and polyendocrine autoimmune disease. Antibodies to GAD in IDDM patients usually recognize conformation-dependent regions on GAD65 and rarely bind to the second isoform, glutamic acid decarboxylase-67 (GAD67). In contrast, those present in stiff man syndrome and polyendocrine disease commonly target the second isoform (GAD67) and include antibodies that are less dependent on the conformation of the molecule. By immortalizing peripheral blood B cells with Epstein-Barr virus, we have generated three human IgG autoantibodies, termed b35, b78, and b96, to GAD65 from one patient with multiple autoantibodies to endocrine organs and Graves' disease. All three autoantibodies are of the IgG1 isotype, with islet cell activity, and do not react with GAD67. The regions on GAD65 recognized by the three autoantibodies have been investigated by immunoprecipitation with a series of chimeras, by binding to denatured and reduced antigens, and using protein footprinting techniques. Using chimeric GAD proteins, we have shown that b35 targets the IDDM-E1 region of GAD65 (amino acids 240-435) whereas both b78 and b96 target the IDDM-E2 region of GAD65 (amino acids 451-570). Furthermore, examination of binding to recombinant GAD65 and GAD67 by Western blotting revealed some differences in epitope recognition, where only b78 bound denatured and reduced GAD65. However, b35, b78, and b96 autoantibodies had different footprinting patterns after trypsin treatment of immune complexes with GAD65, again indicating different epitope recognition. Our results indicate that antibodies to GAD65 present in nondiabetic patients with multiple autoantibodies to endocrine organs show similarities to those in IDDM (by targeting IDDM-E1 and IDDM-E2 regions of GAD65) as well as subtle differences in epitope recognition (such as binding to denatured and reduced GAD65 and by protein footprinting). Thus, the GAD65 epitopes recognized by autoantibodies in different autoimmune diseases may overlap and be more heterogeneous than previously recognized.
A study was made of the effect of ethacizine administered in a single dose and continuously on myocardial contractility and hemodynamics in 78 patients with coronary heart disease, neurocirculatory dystonia and myocardiodystrophy suffering from impaired cardiac rhythm. In 28 patients, ethacizine was administered in combination with glutamic acid and in 23 with digoxin. In patients with coronary heart disease, particularly in those with heart failure, ethacizine produced a negative inotropic effect. Introduction into the treatment of digoxin or glutamic acid smoothed over the cardiodepressive action of the drug. When administered continuously in a dose of 150-300 mg/day, ethacizine is an effective antiarrhythmic drug.
Stiff-man syndrome is a rare disorder of the central nervous system consisting of progressive, fluctuating muscle rigidity with painful spasms. It is occasionally associated with endocrine disorders, including insulin-dependent diabetes, and with epilepsy. We investigated the possible existence of autoimmunity against the nervous system in a patient with stiff-man syndrome associated with epilepsy and Type I diabetes mellitus. Levels of IgG, which had an oligoclonal pattern, were elevated in the cerebrospinal fluid. The serum and the cerebrospinal fluid produced an identical, intense staining of all gray-matter regions when used to stain brain sections according to an indirect light-microscopical immunocytochemical procedure. The staining patterns were identical to those produced by antibodies to glutamic acid decarboxylase (the enzyme responsible for the synthesis of gamma-aminobutyric acid). A band comigrating with glutamic acid decarboxylase in sodium dodecyl sulfate-polyacrylamide gels appeared to be the only nervous-tissue antigen recognized by cerebrospinal fluid antibodies, and the predominant antigen recognized by serum antibodies. These findings support the idea that an impairment of neuronal pathways that operate through gamma-aminobutyric acid is involved in the pathogenesis of stiff-man syndrome, and they raise the possibility of an autoimmune pathogenesis.
Several new antibiotics which contain an N-methyl-thio-tetrazole side chain have been associated with the development of hypoprothrombinaemia. This side chain was found to be capable of the in-vitro inhibition of a necessary reaction in the synthesis of prothrombin, the gamma carboxylation of glutamic acid. The 50% inhibitory concentration was 1.1 mmol/l. The intact antibiotics latamoxef, cephamandole, and cefoperazone, which contain the side chain, did not inhibit the reaction at concentrations up to 2 mmol/l. These results suggest that hypoprothrombinaemia associated with antibiotics containing N-methyl-thio-tetrazole side chains may be a consequence of the in-vivo degradation of these antibiotics and the subsequent inhibition of the gamma carboxylation of glutamic acid by the side chain.