D-aspartate Content Research Articles

DTD1 modulates synaptic efficacy by maintaining D-serine and D-aspartate homeostasis.

D-serine and D-aspartate are involved in N-methyl-D-aspartate receptor (NMDAR)-related physiological and pathological processes. D-aminoacyl-tRNA deacylase 1 (DTD1) may biochemically contribute to D-serine or D-aspartate production. However, it is unclear thus far whether DTD1 regulates D-serine or D-aspartate content in neurobiological processes. In the present research, we found that DTD1 was essential to maintain the D-serine or D-aspartate homeostasis, which was consistent with the phenomenon that DTD1-deficiency resulted in changes in the quantity changes of functional NMDAR subunits in postsynaptic compartments. Moreover, DTD1 played a considerable role in regulating dendritic morphology and synaptic structure. As a consequence, DTD1 affected neurobiological events, including the synaptic strength of the CA3-to-CA1 circuit, dendritic spine density of hippocampal pyramidal neurons, and behavioral performance of mice in the Morris water maze. These findings highlight the important role of DTD1 in synaptic transmission, neuronal morphology, and spatial learning and memory and suggest an undisclosed mechanism of DTD1 that participates the regulation of D-serine or D-aspartate homeostasis in hippocampal neurons.

Dysfunctional d-aspartate metabolism in BTBR mouse model of idiopathic autism

BackgroundAutism spectrum disorders (ASD) comprise a heterogeneous group of neurodevelopmental conditions characterized by impairment in social interaction, deviance in communication, and repetitive behaviors. Dysfunctional ionotropic NMDA and AMPA receptors, and metabotropic glutamate receptor 5 activity at excitatory synapses has been recently linked to multiple forms of ASD. Despite emerging evidence showing that d-aspartate and d-serine are important neuromodulators of glutamatergic transmission, no systematic investigation on the occurrence of these D-amino acids in preclinical ASD models has been carried out. MethodsThrough HPLC and qPCR analyses we investigated d-aspartate and d-serine metabolism in the brain and serum of four ASD mouse models. These include BTBR mice, an idiopathic model of ASD, and Cntnap2−/−, Shank3−/−, and 16p11.2+/− mice, three established genetic mouse lines recapitulating high confidence ASD-associated mutations. ResultsBiochemical and gene expression mapping in Cntnap2−/−, Shank3−/−, and 16p11.2+/− failed to find gross cerebral and serum alterations in d-aspartate and d-serine metabolism. Conversely, we found a striking and stereoselective increased d-aspartate content in the prefrontal cortex, hippocampus and serum of inbred BTBR mice. Consistent with biochemical assessments, in the same brain areas we also found a robust reduction in mRNA levels of d-aspartate oxidase, encoding the enzyme responsible for d-aspartate catabolism. ConclusionsOur results demonstrated the presence of disrupted d-aspartate metabolism in a widely used animal model of idiopathic ASD. General significanceOverall, this work calls for a deeper investigation of D-amino acids in the etiopathology of ASD and related developmental disorders.

Prenatal expression of D-aspartate oxidase causes early cerebral D-aspartate depletion and influences brain morphology and cognitive functions at adulthood.

The free D-amino acid, D-aspartate, is abundant in the embryonic brain but significantly decreases after birth. Besides its intracellular occurrence, D-aspartate is also present at extracellular level and acts as an endogenous agonist for NMDA and mGlu5 receptors. These findings suggest that D-aspartate is a candidate signaling molecule involved in neural development, influencing brain morphology and behaviors at adulthood. To address this issue, we generated a knockin mouse model in which the enzyme regulating D-aspartate catabolism, D-aspartate oxidase (DDO), is expressed starting from the zygotic stage, to enable the removal of D-aspartate in prenatal and postnatal life. In line with our strategy, we found a severe depletion of cerebral D-aspartate levels (up to 95%), since the early stages of mouse prenatal life. Despite the loss of D-aspartate content, Ddo knockin mice areviable, fertile, and show normal gross brain morphology at adulthood. Interestingly, early D-aspartate depletion is associated with a selective increase in the number of parvalbumin-positive interneurons in the prefrontal cortex and also with improved memory performance in Ddo knockin mice. In conclusion, the present data indicate for the first time a biological significance of precocious D-aspartate in regulating mouse brain formation and function at adulthood.

The Emerging Role of Altered d-Aspartate Metabolism in Schizophrenia: New Insights From Preclinical Models and Human Studies.

Besides d-serine, another d-amino acid with endogenous occurrence in the mammalian brain, d-aspartate, has been recently shown to influence NMDA receptor (NMDAR)-mediated transmission. d-aspartate is present in the brain at extracellular level in nanomolar concentrations, binds to the agonist site of NMDARs and activates this subclass of glutamate receptors. Along with its direct effect on NMDARs, d-aspartate can also evoke considerable l-glutamate release in specific brain areas through the presynaptic activation of NMDA, AMPA/kainate and mGlu5 receptors. d-aspartate is enriched in the embryonic brain of rodents and humans and its concentration strongly decreases after birth, due to the post-natal expression of the catabolising enzyme d-aspartate oxidase (DDO). Based on the hypothesis of NMDAR hypofunction in schizophrenia pathogenesis, recent preclinical and clinical studies suggested a relationship between perturbation of d-aspartate metabolism and this psychiatric disorder. Consistently, neurophysiological and behavioral characterization of Ddo knockout (Ddo−/−) and d-aspartate-treated mice highlighted that abnormally higher endogenous d-aspartate levels significantly increase NMDAR-mediated synaptic plasticity, neuronal spine density and memory. Remarkably, increased d-aspartate levels influence schizophrenia-like phenotypes in rodents, as indicated by improved fronto-hippocampal connectivity, attenuated prepulse inhibition deficits and reduced activation of neuronal circuitry induced by phencyclidine exposure. In healthy humans, a genetic polymorphism associated with reduced prefrontal DDO gene expression predicts changes in prefrontal phenotypes including greater gray matter volume and enhanced functional activity during working memory. Moreover, neurochemical detections in post-mortem brain of schizophrenia-affected patients have shown significantly reduced d-aspartate content in prefrontal regions, associated with increased DDO mRNA expression or DDO enzymatic activity. Overall, these findings suggest a possible involvement of dysregulated embryonic d-aspartate metabolism in schizophrenia pathophysiology and, in turn, highlight the potential use of free d-aspartate supplementation as a new add-on therapy for treating the cognitive symptoms of this mental illness.

Structure–function relationships in human d-aspartate oxidase: characterisation of variants corresponding to known single nucleotide polymorphisms

d-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for the acidic amino acid d-aspartate, an endogenous agonist of the N-methyl-d-aspartate (NMDA) receptor. Dysregulation of NMDA receptor-mediated neurotransmission has been implicated in the onset of various neuropsychiatric disorders including schizophrenia and in chronic pain. Thus, appropriate regulation of the amount of d-aspartate is believed to be important for maintaining proper neural activity in the nervous system. Herein, the effects of the non-synonymous single nucleotide polymorphisms (SNPs) R216Q and S308N on several properties of human DDO were examined. Analysis of the purified recombinant enzyme showed that the R216Q and S308N substitutions reduce enzyme activity towards acidic d-amino acids, decrease the binding affinity for the coenzyme flavin adenine dinucleotide and decrease the temperature stability. Consistent with these findings, further experiments using cultured mammalian cells revealed elevated d-aspartate in cultures of R216Q and S308N cells compared with cells expressing wild-type DDO. Furthermore, accumulation of several amino acids other than d-aspartate also differed between these cultures. Thus, expression of DDO genes carrying the R216Q or S308N SNP substitutions may increase the d-aspartate content in humans and alter homeostasis of several other amino acids. This work may aid in understanding the correlation between DDO activity and the risk of onset of NMDA receptor-related diseases.

Decreased free d-aspartate levels are linked to enhanced d-aspartate oxidase activity in the dorsolateral prefrontal cortex of schizophrenia patients

It is long acknowledged that the N-methyl d-aspartate receptor co-agonist, d-serine, plays a crucial role in several N-methyl d-aspartate receptor-mediated physiological and pathological processes, including schizophrenia. Besides d-serine, another free d-amino acid, d-aspartate, is involved in the activation of N-methyl d-aspartate receptors acting as an agonist of this receptor subclass, and is abundantly detected in the developing human brain. Based on the hypothesis of N-methyl d-aspartate receptor hypofunction in the pathophysiology of schizophrenia and considering the ability of d-aspartate and d-serine to stimulate N-methyl d-aspartate receptor-dependent transmission, in the present work we assessed the concentration of these two d-amino acids in the post-mortem dorsolateral prefrontal cortex and hippocampus of patients with schizophrenia and healthy subjects. Moreover, in this cohort of post-mortem brain samples we investigated the spatiotemporal variations of d-aspartate and d-serine. Consistent with previous work, we found that d-aspartate content was selectively decreased by around 30% in the dorsolateral prefrontal cortex, but not in the hippocampus, of schizophrenia-affected patients, compared to healthy subjects. Interestingly, such selective reduction was associated to greater (around 25%) cortical activity of the enzyme responsible for d-aspartate catabolism, d-aspartate oxidase. Conversely, no significant changes were found in the methylation state and transcription of DDO gene in patients with schizophrenia, compared to control individuals, as well as in the expression levels of serine racemase, the major enzyme responsible for d-serine biosynthesis, which also catalyzes aspartate racemization. These results reveal the potential involvement of altered d-aspartate metabolism in the dorsolateral prefrontal cortex as a factor contributing to dysfunctional N-methyl d-aspartate receptor-mediated transmission in schizophrenia.

Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging.

The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate.

Identification and characterization of natural microbial products that alter the free d-aspartate content of mammalian cells.

Mammalian cells possess the molecular apparatus necessary to take up, degrade, synthesize, and release free d-aspartate, which plays an important role in physiological functions within the body. Here, biologically active microbial compounds and pre-existing drugs were screened for their ability to alter the intracellular d-aspartate level in mammalian cells, and several candidate compounds were identified. Detailed analytical studies suggested that two of these compounds, mithramycin A and geldanamycin, suppress the biosynthesis of d-aspartate in cells. Further studies suggested that these compounds act at distinct sites within the cell. These compounds may advance our current understanding of biosynthesis of d-aspartate in mammals, a whole picture of which remains to be disclosed.

D-Aspartate: An endogenous NMDA receptor agonist enriched in the developing brain with potential involvement in schizophrenia

Free d-aspartate and d-serine occur at substantial levels in the mammalian brain. d-Serine is a physiological endogenous co-agonist for synaptic N-Methyl d-Aspartate (NMDA) receptors (NMDARs), and is involved in the pathophysiology of schizophrenia. Much less is known about the biological meaning of d-aspartate. d-Aspartate is present at high levels in the embryo brain and strongly decreases at post-natal phases. Temporal reduction of d-aspartate levels depends on the post-natal onset of d-aspartate oxidase (DDO), an enzyme able to selectively catabolize this d-amino acid. Pharmacological evidence indicates that d-aspartate binds to and activates NMDARs. Characterization of genetic and pharmacological mouse models with abnormally higher levels of d-aspartate has evidenced that increased d-aspartate enhances hippocampal NMDAR-dependent synaptic plasticity, dendritic morphology and spatial memory. In line with the hypothesis of a hypofunction of NMDARs in the pathogenesis of schizophrenia, it has been shown that increased d-aspartate levels also improve brain connectivity, produce corticostriatal adaptations resembling those observed after chronic haloperidol treatment, and protects against prepulse inhibition deficits and abnormal circuits activation induced by psychotomimetic drugs. In healthy humans, genetic variation predicting reduced expression of DDO in post-mortem prefrontal cortex is associated with greater prefrontal gray matter and activity during working memory. On the other side, evaluation of d-aspartate content in post-mortem patients with schizophrenia has shown a significant reduction of this d-amino acid in the prefrontal cortex and striatum. Generation of mouse models with reduced embryonic levels of d-aspartate may disclose unprecedented role for d-aspartate in developmental brain processes associated with vulnerability to psychotic-like symptoms.

The free D-Aspartic acid and D-Glumatic acid content of sheep milk and sheep milk products

The role of D-amino acids of foods in the human health is a strongly discussed topic and usually, data came from the investigation of cow milk. We have studied the free D-aspartic acid and free D-glutamic acid content of sheep milk, heat-treated sheep milk at various temperatures and various products of sheep milk. Raw sheep milk didn’t contain free D-aspartic and D-glutamic acid in remarkable amount and ratio 5.92% free D-aspartic acid; 2.62% free D-glutamic acid). Our heat-treatments didn’t cause major change in the free D-aspartic and D-glutamic acid content (max.: 7.8% free D-aspartic acid; 5.3% free D-glutamic acid in total free aspartic and glutamic acid). Contrary, all of the investigated products contained high level of free D-amino acids. The free D-aspartic acid and free D-glutamic acid content of the products were 16,9-39,5%,and 13,3-27% in the percent of total free amino acids. The racemization of aspartic acid was higher, than that of glutamic acid in every product. The D-amino acid content of fermented milk products was higher than in different cheeses.

Decreased levels of d-aspartate and NMDA in the prefrontal cortex and striatum of patients with schizophrenia

The potential implication of a decrease in the function of N-methyl-d-aspartate receptors (NMDARs) in the pathophysiology of schizophrenia has long been hypothesised. Accordingly, compounds that inhibit the glycine-1 transporter or target the glycine-binding site of NMDARs, including the co-agonists d-serine and glycine, have shown promise in treating the symptoms of schizophrenia. Clinical interest for d-serine has also been supported by evidence for its abnormal metabolism in schizophrenic patients. Together with d-serine, another d-form amino acid, d-aspartate, exists in the brain of mammals. Synthesised by the enzyme aspartate racemase, d-aspartate is highly concentrated in the prenatal brain; after birth, its levels sharply decrease due to the catabolising activity of the enzyme d-aspartate oxidase. d-aspartate is able to stimulate NMDAR-dependent neurotransmission through direct action at the glutamate-binding site of NMDARs, thus functioning as an endogenous agonist for this subclass of glutamate receptors. In this study, we evaluated for the first time the content of d-aspartate and of its derivative, NMDA, in the post-mortem prefrontal cortex and striatum of schizophrenic patients. Moreover, in the same brain samples, we analysed the expression levels of the subunits that form NMDARs, which are the in vivo targets of d-aspartate and NMDA. Interestingly, we found that d-aspartate and NMDA are consistently decreased in schizophrenia brains compared to control brains. In the prefrontal cortex, this decrease is correlated with a marked downregulation of NMDAR subunits. Overall, these results agree with the innovative therapeutic research in schizophrenia that is aimed at targeting glutamatergic transmission viad-amino acids.

Increased levels of d-aspartate in the hippocampus enhance LTP but do not facilitate cognitive flexibility

In the present study, we demonstrate a direct role for d-aspartate in regulating hippocampal synaptic plasticity. These evidences were obtained using two different experimental strategies which enabled a non-physiological increase of endogenous d-aspartate levels in the mouse hippocampus: a genetic approach based on the targeted deletion of d-aspartate oxidase gene and another based on the oral administration of d-aspartate. Overall, our results indicate that increased d-aspartate content does not affect basal properties of synaptic transmission but enhances long-term potentiation in hippocampal slices from both genetic and pharmacological animal models. Besides electrophysiological data, behavioral analysis suggests that altered levels of d-aspartate in the hippocampus do not perturb basal spatial learning and memory abilities, but may selectively interfere with the dynamic NMDAR-dependent processes underlying cognitive flexibility.

Accumulation of altered aspartyl residues in erythrocyte proteins from patients with Down's syndrome

Spontaneous protein deamidation of labile Asn residues, generating L-isoaspartates and D-aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be 'repaired' by a specific L-isoaspartate (D-aspartate) protein O-methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Down's syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age-matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of L-isoaspartates in erythrocyte membrane proteins from DS patients. The content of D-aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl-labeled methionine; a three-fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein-Barr virus B-lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.

D‐Aspartate as a putative cell–cell signaling molecule in the Aplysia californica central nervous system

The content, synthesis and transport of D-aspartate (D-Asp) in the CNS of Aplysia californica is investigated using capillary electrophoresis (CE) with both laser-induced fluorescence and radionuclide detection. Millimolar concentrations of D-Asp are found in various regions of the CNS. In the cerebral ganglion, three adjacent neuronal clusters have reproducibly different D-Asp levels; for example, in the F- and C-clusters, up to 85% of the free Asp is present in the D-form. Heterogeneous distribution of D-Asp is also found in the individual identified neurons tested, including the optical ganglion top-layer neurons, metacerebral cells, R2 neurons, and F-, C- and G-cluster neurons. The F-cluster neurons have the highest percentage of D-Asp (approximately 58% of the total Asp), whereas the lowest value of approximately 8% is found in R2 neurons. In pulse-chase experiments with radiolabeled D-Asp, followed by CE with radionuclide detection, the synthesis of D-Asp from L-aspartate (L-Asp) is confirmed. Is D-Asp in the soma, or is it transported to distantly located release sites? D-Asp is clearly detected in the major nerves of A. californica, including the pleuroabdominal and cerebrobuccal connectives and the anterior tentacular nerves, suggesting it is transported long distances. In addition, both D-Asp and L-Asp are transported in the pleuroabdominal connectives in a colchicine-dependent manner, whereas several other amino acids are not. Finally, d-Asp produces electrophysiological effects similar to those induced by L-Asp. These data are consistent with an active role for D-Asp in cell-to-cell communication.

Protein L-Isoaspartyl Methyltransferase Catalyzes in Vivo Racemization of Aspartate-25 in Mammalian Histone H2B

Protein L-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical L-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the D/L ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp25 (the major PIMT target site in H2B) was significantly racemized, exhibiting d/l ratios as high as 0.12, whereas Asp51, a comparison site, exhibited negligible racemization (D/L < or = 0.01). Racemization of Asp25 was independent of animal age over the range of 2-15 years. Using H2B from 2-3-week mouse brain, we found a similar D/L ratio (0.14) at Asp25 in wild type mice, but substantially less racemization (D/L = 0.035) at Asp25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that D-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age.

D-aspartic acid induces merocrine secretion in the frog harderian gland

High levels of D-aspartic acid (D-Asp) have been found in the harderian gland (hg) of the frog,Rana esculenta. This is the first report of D-aspartate in an exocrine gland. D-aspartate content is correlated with secretory activity: it is high in July when the hg shows the highest secretory activity, lower in February, in concomitance with the low secretory activity. The harderian gland has the capacity to uptake D-Asp injected i.p. In July, gland uptake is higher than in February. Administration of 2.0 µmol/g D-Asp in frogs during both periods induces the release of secretory granules in the hg. This effect is more evident in July, when the amino acid accumulates at the apex of the cells beneath the plasma membrane. Such evidence suggests a role of D-Asp in the exocytotic mechanism.

Aging at the molecular level exemplified by proteins

During their life span, human proteins may undergo post-translational modifications that can be interpreted as manifestations of protein aging. The most frequently discussed modifications in that context are oxidation, glycation, deamidation, isomerization and racemization. Their pathophysiological relevance depends on the availability of repair mechanisms and on the protein turnover. Proteins with high turnover will be exchanged before molecular modifications become relevant. The most affected proteins are long-living and permanent proteins. Such proteins can be identified by the determination of their D-aspartic content (as a measure of an in vivo racemization and isomerization of aspartyl and asparaginyl residues). Using this method it could be demonstrated that numerous proteins in diverse human tissues are long-living or permanent and cannot escape post-translational modification by turnover. The human organism is confronted with an accumulation of "abnormal", post-translationally modified proteins during aging, especially in the extracellular space, but also at a cellular level. The exact pathophysiological dimension of post-translational modifications as manifestations of protein aging has to be further elucidated. However, they have been already discussed as relevant factors in the pathogenesis of diseases of old age.

D-Aspartate oxidase and free acidic d-amino acids in lower vertebrates

d-Aspartate oxidase and free d-aspartate and/or d-glutamate have been found in various tissues of lower vertebrates including avians, amphibians and fishes. The enzymes of those animals were similar to the mammalian enzymes in substrate and inhibitor specificity. N-Methyl- d-aspartate is the best substrate of the enzymes of those animals, followed by d-aspartate, meso-2,3-diaminosuccinate and d-glutamate. In avians and amphibians, the highest activity was found in the kidneys, whereas, in fishes, hepatic activity was equal to or greater than renal activity. Male chickens had significantly higher activities in the liver, kidneys and pancreas than female. However, no significant sex difference was observed in other animals. Substantial amounts of d-aspartate and/or d-glutamate were detected in all tissues examined, irrespective of species. With the exception of the spleen or testes of some species, the d-glutamate content was equal to or greater than the d-aspartate content. The amounts of d-enantiomer present were significantly different between two sexes in several tissues of avians and amphibians. However, no common relationship was observed between the d-enantiomer contents and d-aspartate oxidase activity. The involvement of d-aspartate in the control of androgen secretion by the ovary was demonstrated in a green frog.

Occurrence of peptidyl d-amino acids in soluble fractions of several eubacteria, archaea and eukaryotes

The occurrence of peptidyl d-amino acids in the aqueous soluble fractions was investigated in various eubacteria, some archaea and some eukaryotes. The contents of the d-enantiomers of serine, alanine, proline, glutamate (glutamine), aspartate (asparagine) and phenylalanine were determined with cell- and tissue-extracts, by means of acid hydrolysis and high-performance liquid chromatography. The rate of d-enantiomer (%, the ratio in molar concentration of a d-amino acid to the total of the d-amino acid and the corresponding l-amino acid) of alanine and glutamate were high in some Gram-positive eubacteria: 11.7% in Staphylococcus epidermidis and 10.3% in Streptococcus pyogenes for alanine, and 22.3% for glutamate in Bacillus YN-1. The d-glutamate content was also high (8.0%) in the Gram-negative eubacterium, Thiobacillus ferrooxidans. d-Aspartate was common, as was d-glutamate: the highest d-aspartate content was detected in an archaeum, Pyrobaculum islandicum (4.0%). However, the content of d-aspartate was low, 0.2–1.8% in most other bacteria. The presence of d-serine was shown in some organisms, but that of d-proline was scarce. The d-enantiomer of phenylalanine was not detected in any of the organisms examined. These results indicate that of the bacteria examined herein most Gram-negative and some Gram-positive eubacteria, as well as archaea contain only low levels of d-amino acids in the soluble peptidyl fraction, and the levels were comparable to those in eukaryotes examined. To our knowledge, the general presence of peptidyl d-amino acids in these organisms, especially archaea and eukaryotic cells including those from rat liver tissues, has been shown here for the first time.

Net glutamate release from astrocytes is not induced by extracellular potassium concentrations attainable in brain.

Elevated extracellular potassium concentration ([K+]e) has been shown to induce reversal of glial Na+-dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K+]e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K+]e (by equimolar substitution of K+ for Na+), and glutamate accumulation was measured by HPLC. With [K+]e elevations to 60 mM, medium glutamate concentrations did not increase during incubation periods of 5-120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50-100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer D-aspartate, and parallel results were obtained; no increase in medium D-aspartate content resulted from [K+]e elevation up to 90 mM, whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K+]e attainable in brain.