Glutamate decarboxylase is not genetically linked to pyridoxine-dependent seizures.

Several aspects of pyridoxine-dependent seizure (PDS) suggest a mutation affecting glutamate decarboxylase (GAD) as a possible cause. To examine the possibility of GAD linkage with PDS, the authors performed genotype analyses of three families using polymorphic markers near the GAD genes (GAD1 and GAD2). In each family, the affected siblings exhibited different genotypes for the GAD2 gene; in two families the GAD1 genotype was disparate. These findings suggest that a mutation of GAD is not directly involved in all cases of PDS.

Glutamine stimulates gamma-aminobutyric acid synthesis in synaptosomes but other putative astrocyte-to-neuron shuttle substrates do not.

GABAergic neurons require a supply of precursor glutamate for gamma-aminobutyric acid (GABA) synthesis to maintain their GABA levels. Because neurons lack the anaplerotic enzymes necessary for net synthesis of glutamate from glucose, they depend on astrocytes to supply compounds that can be metabolized to glutamate and ultimately used for GABA production. To test the effect of putative astrocytic shuttle metabolites on GABA synthesis, we used synaptosomes prepared from substantia nigra, an area rich in GABAergic terminals. The low number of glutamatergic endings in the nigral preparation allows a more accurate measurement of glutamate present in GABAergic endings. GABA synthesis by nigral synaptosomes was stimulated 3.1-fold when 500 microM glutamine was added to the incubation medium. Glutamate amounts also increased. In contrast, the possible precursor metabolites. 2-oxoglutarate (2-OG), malate and citrate, failed to stimulate GABA synthesis over the rate observed with control medium. Unlike malate and citrate. 2-OG reduced the decline in total glutamate observed when synaptosomes were incubated in control. In contrast to glutamine the production of synaptosomal glutamate from 2-OG, malate, and citrate is not great enough to stimulate GABA synthesis.

Synaptosomal glutamate uptake declines progressively in the spinal cord of a mutant mouse with motor neuron disease.

It has been suggested that the degeneration of motor neurons in amyotrophic lateral sclerosis is a consequence of excitotoxicity resulting from a loss of synaptosomal glutamate uptake. The role of synaptosomal glutamate uptake in the pathogenesis of motor neuron disease was studied in the Mnd mouse. Glutamate uptake in spinal-cord synaptosomes declined in parallel with the onset of behavioral deficits in Mnd mice but lagged considerably behind the appearance of pathology in motor neurons. Glutamate uptake did not decline significantly in corpus striatum, and GABA uptake did not change significantly in either spinal cord or striatum. The presence of pronounced histopathological changes before the loss of glutamate uptake suggests that the decline of glutamate uptake is a consequence rather than the primary cause of motor neuron disease in the Mnd mouse.

GABA synthesis in brain slices is dependent on glutamine produced in astrocytes.

The rate of gamma-aminobutyric acid (GABA) synthesis in rat-brain slices was determined by inhibiting GABA transaminase with 20-microM gabaculine and measuring the increase of GABA. Added 500-microM glutamine increased the rate of GABA synthesis by 50%, indicating that glutamate decarboxylase is not saturated in brain slices. The stimulation of GABA synthesis with added glutamine in brain slices was much less than that reported for synaptosomes. The lower stimulation in slices was attributable to astrocytic glutamine production, as the rate of GABA synthesis decreased by 44% when glutamine production was inhibited with methionine sulfoximine. Added glutamine restored the rate to the maximal value observed in brain slices. The rate of GABA synthesis was decreased by 65% in slices pretreated with an inhibitor of glutaminase, and added glutamine did not reverse this effect. These results suggest that glutamine produced by astrocytes is a quantitatively important precursor of GABA synthesis in cortical slices.

Stimulation of synaptosomal gamma-aminobutyric acid synthesis by glutamate and glutamine.

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.

Gono Gen coagglutination test for confirmation of Neisseria gonorrhoeae.

The Gono Gen (Micro-Media Systems, Inc., Potomac, Md.) coagglutination test was compared with the sugar utilization test and with a direct fluorescent antibody test for confirmation of Neisseria gonorrhoeae. Of 110 gonococcal clinical isolates, 109 were positive by the Gono Gen test. Of 57 nongonococcal gram-negative diplococci, all were negative by the Gono Gen test. We conclude that the Gono Gen test is sensitive and highly specific and provides a rapid method for the confirmation of N. gonorrhoeae.