Covalent linkage of myristic acid to the N-terminal glycine residue of Pr55gag, the precursor of the major structural proteins of human immunodeficiency virus 1 (HIV-1), facilitates an essential step in virus assembly and propagation. Substitution of the myristoyl-acceptor glycine with alanine, in a functional clone of HIV-1, eliminates virus replication. Complementation of this defect, in trans, restores infectious particle production. The nonmyristoylated (myr-) gag precursor accumulates in infected cells and is not processed into the mature capsid components of the intact virion. However, myr- Pr55gag can be processed by purified HIV protease in vitro, demonstrating that the myristoyl moiety is not required for cleavage by the protease. Myristoylation of Pr55gag is not necessary for localization but is required for stable membrane association and assembly of HIV-1.

In an attempt to account for antibody specificity and complementarity in terms of structure, human kappa-, human lambda-, and mouse kappa-Bence Jones proteins and light chains are considered as a single population and the variable and constant regions are compared using the sequence data available. Statistical criteria are used in evaluating each position in the sequence as to whether it is essentially invariant or group-specific, subgroup-specific, species-specific, etc. Examination of the invariant residues of the variable and constant regions confirms the existence of a large number of invariant glycines, no invariant valine, lysine, and histidine, and only one invariant leucine and alanine in the variable region, as compared with the absence of invariant glycines and presence of three each of invariant alanine, leucine, and valine and two each of invariant lysine and histidine in the constant region. The unique role of glycine in the variable region is emphasized. Hydrophobicity of the invariant residues of the two regions is also evaluated. A parameter termed variability is defined and plotted against the position for the 107 residues of the variable region. Three stretches of unusually high variability are noted at residues 24-34, 50-56, and 89-97; variations in length have been found in the first and third of these. It is hypothesized that positions 24-34 and 89-97 contain the complementarity-determining residues of the light chain-those which make contact with the antigenic determinant. The heavy chain also has been reported to have a similar region of very high variability which would also participate in forming the antibody-combining site. It is postulated that the information for site complementarity is contained in some extrachromosomal DNA such as an episome and is incorporated by insertion into the DNA of the structural genes for the variable region of short linear sequences of nucleotides. The advantages and disadvantages of this hypothesis are discussed.

The integrins are a superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. They are transmembrane alphabeta heterodimers and at least 18 alpha and eight beta subunits are known in humans, generating 24 heterodimers. Members of this family have been found in mammals, chicken and zebrafish, as well as lower eukaryotes, including sponges, the nematode Caenorhabditis elegans (two alpha and one beta subunits, generating two integrins) and the fruitfly Drosophila melanogaster (five alpha and one beta, generating five integrins). The alpha and beta subunits have distinct domain structures, with extracellular domains from each subunit contributing to the ligand-binding site of the heterodimer. The sequence arginine-glycine-aspartic acid (RGD) was identified as a general integrin-binding motif, but individual integrins are also specific for particular protein ligands. Immunologically important integrin ligands are the intercellular adhesion molecules (ICAMs), immunoglobulin superfamily members present on inflamed endothelium and antigen-presenting cells. On ligand binding, integrins transduce signals into the cell interior; they can also receive intracellular signals that regulate their ligand-binding affinity. Here we provide a brief overview that concentrates mostly on the organization, structure and function of mammalian integrins, which have been more extensively studied than integrins in other organisms.

Semiconductor quantum dots (QDs) are nanometer-sized fluorescent probes suitable for advanced biological imaging. We used QDs to track individual glycine receptors (GlyRs) and analyze their lateral dynamics in the neuronal membrane of living cells for periods ranging from milliseconds to minutes. We characterized multiple diffusion domains in relation to the synaptic, perisynaptic, or extrasynaptic GlyR localization. The entry of GlyRs into the synapse by diffusion was observed and further confirmed by electron microscopy imaging of QD-tagged receptors.

Central sensitization, increased sensitivity in spinal cord dorsal horn neurons after injuries, plays an essential role in the induction and maintenance of chronic pain. However, synaptic mechanisms underlying central sensitization are incompletely known. Growing evidence suggests that proinflammatory cytokines (PICs), such as interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha), are induced in the spinal cord under various injury conditions and contribute to pain hypersensitivity. Using patch-clamp recordings in lamina II neurons of isolated spinal cord slices, we compared the effects of IL-1beta, IL-6, and TNFalpha on excitatory and inhibitory synaptic transmission. Whereas TNFalpha enhanced the frequency of spontaneous EPSCs (sEPSCs), IL-6 reduced the frequency of spontaneous IPSCs (sIPSCs). Notably, IL-1beta both enhanced the frequency and amplitude of sEPSCs and reduced the frequency and amplitude of sIPSCs. Consistently, TNFalpha and IL-1beta enhanced AMPA- or NMDA-induced currents, and IL-1beta and IL-6 suppressed GABA- and glycine-induced currents. Furthermore, all the PICs increased cAMP response element-binding protein (CREB) phosphorylation in superficial dorsal horn neurons and produced heat hyperalgesia after spinal injection. Surprisingly, soluble IL-6 receptor (sIL-6R) produced initial decrease of sEPSCs, followed by increase of sEPSCs and CREB phosphorylation. Spinal injection of sIL-6R also induced heat hyperalgesia that was potentiated by coadministration with IL-6. Together, our data have demonstrated that PICs induce central sensitization and hyperalgesia via distinct and overlapping synaptic mechanisms in superficial dorsal horn neurons either by increasing excitatory synaptic transmission or by decreasing inhibitory synaptic transmission. PICs may further induce long-term synaptic plasticity through CREB-mediated gene transcription. Blockade of PIC signaling could be an effective way to suppress central sensitization and alleviate chronic pain.

Long-term potentiation (LTP) of excitatory transmission in the hippocampus likely contributes to learning and memory. The mechanisms underlying LTP at these synapses are not well understood, although phosphorylation and redistribution of AMPA receptors may be responsible for this form of synaptic plasticity. We show here that miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons reliably demonstrate LTP when postsynaptic NMDA receptors are briefly stimulated with glycine. LTP of these synapses is accompanied by a rapid insertion of native AMPA receptors and by increased clustering of AMPA receptors at the surface of dendritic membranes. Both LTP and glycine-facilitated AMPA receptor insertion are blocked by intracellular tetanus toxin (TeTx), providing evidence that AMPA receptors are inserted into excitatory synapses via a SNARE-dependent exocytosis during LTP.

DNA analogues are currently being intensely investigated owing to their potential as gene-targeted drugs. Furthermore, their properties and interaction with DNA and RNA could provide a better understanding of the structural features of natural DNA that determine its unique chemical, biological and genetic properties. We recently designed a DNA analogue, PNA, in which the backbone is structurally homomorphous with the deoxyribose backbone and consists of N-(2-aminoethyl)glycine units to which the nucleobases are attached. We showed that PNA oligomers containing solely thymine and cytosine can hybridize to complementary oligonucleotides, presumably by forming Watson-Crick-Hoogsteen (PNA)2-DNA triplexes, which are much more stable than the corresponding DNA-DNA duplexes, and bind to double-stranded DNA by strand displacement. We report here that PNA containing all four natural nucleobases hybridizes to complementary oligonucleotides obeying the Watson-Crick base-pairing rules, and thus is a true DNA mimic in terms of base-pair recognition.

The crystal structure has been determined at 2.8 A resolution for a chemically-trapped covalent reaction intermediate between the HhaI DNA cytosine-5-methyltransferase, S-adenosyl-L-homocysteine, and a duplex 13-mer DNA oligonucleotide containing methylated 5-fluorocytosine at its target. The DNA is located in a cleft between the two domains of the protein and has the characteristic conformation of B-form DNA, except for a disrupted G-C base pair that contains the target cytosine. The cytosine residue has swung completely out of the DNA helix and is positioned in the active site, which itself has undergone a large conformational change. The DNA is contacted from both the major and the minor grooves, but almost all base-specific interactions between the enzyme and the recognition bases occur in the major groove, through two glycine-rich loops from the small domain. The structure suggests how the active nucleophile reaches its target, directly supports the proposed mechanism for cytosine-5 DNA methylation, and illustrates a novel mode of sequence-specific DNA recognition.

Buthionine sulfoximine (S-n-butyl homocysteine sulfoximine), the most potent of a series of analogs of methionine sulfoximine thus far studied (Griffith, O.W., Anderson, M.E., and Meister, A. (1979) J. Biol. Chem. 254, 1205-1210), inhibited gamma-glutamylcysteine synthetase about 20 times more effectively than did prothionine sulfoximine and at least 100 times more effectively than methionine sulfoximine. The findings support the conclusion that the S-alkyl moiety of the sulfoximine binds at the enzyme site that normally binds the acceptor amino acid. Thus, the affinity of the enzyme for the S-ethyl, S-n-propyl, and S-n-butyl sulfoximines increases in a manner which is parallel to those of the corresponding isosteric acceptor amino acid substrates, i.e. glycine, alanine, and alpha-aminobutyrate. Buthionine sulfoximine did not inhibit glutamine synthetase detectably, nor did it produce convulsions when injected into mice. Injection of buthionine sulfoximine into mice decreased the level of glutathione in the kidney to a greater extent (less than 20% of the control level) than found previously after giving prothionine sulfoximine. alpha-Methyl buthionine sulfoximine was also prepared and found to be almost as effective as buthionine sulfoximine; this compound would not be expected to undergo substantial degradative metabolism. Buthionine sulfoximine and alpha-methyl buthionine sulfoximine may be useful agents for inhibition of glutathione synthesis in various experimental systems.

: 1. After 50 years of antipsychotic drug development focused on the dopamine D2 receptor, schizophrenia remains a chronic, disabling disorder for most affected individuals. 2. Studies over the last decade demonstrate that administration of low doses of NMDA receptor antagonists can cause in normal subjects the negative symptoms, cognitive impairments and physiologic disturbances observed in schizophrenia. 3. Furthermore, a number of recently identified risk genes for schizophrenia affect NMDA receptor function or glutamatergic neurotransmission. 4. Placebo-controlled trials with agents that directly or indirectly activate the glycine modulatory site on the NMDA receptor have shown reduction in negative symptoms, improvement in cognition and in some cases reduction in positive symptoms in schizophrenic patients receiving concurrent antipsychotic medications. 5. Thus, hypofunction of the NMDA receptor, possibly on critical GABAergic inter-neurons, may contribute to the pathophysiology of schizophrenia.

The receptor specificity of 56 H2 and H3 influenza virus isolates from various animal species has been determined to test the relevance of receptor specificity to the ecology of influenza virus. The results show that the receptor specificity of both H2 and H3 isolates evaluated for sialic acid linkage specificity and inhibition of hemagglutination by horse serum correlates with the species of origin, as postulated earlier for H3 strains based on a limited survey of five human, three avian, and one equine strain. Elucidation of the amino acid sequence of several human H2 receptor variants and analysis of known sequences of H2 and H3 isolates revealed that receptor specificity varies in association with an amino acid change at residues 228 in addition to the change at residue 226 previously documented to affect receptor specificity of H3 but not H1 isolates. Residues 226 and 228 are leucine and serine in human isolates, which preferentially bind sialic acid alpha 2,6-galactose beta 1,4-N-acetyl glucosamine (SA alpha 2,6Gal), and glutamine and glycine in avian and equine isolates, which exhibit specificity for sialic acid alpha-2,3-galactose beta-1,3-N-acetyl galactosamine (SA alpha 2,3Gal). The results demonstrate that the correlation of receptor specificity and species of origin is maintained across both H2 and H3 influenza virus serotypes and provide compelling evidence that influenza virus hosts exert selective pressure to maintain the receptor specificity characteristics of strains isolated from that species.

NMDA receptor antagonists block conditioned fear extinction when injected systemically and also when infused directly into the amygdala. Here we evaluate the ability of D-cycloserine (DCS), a partial agonist at the strychnine-insensitive glycine-recognition site on the NMDA receptor complex, to facilitate conditioned fear extinction after systemic administration or intra-amygdala infusions. Rats received 10 pairings of a 3.7 sec light and a 0.4 mA footshock (fear conditioning). Fear-potentiated startle (increased startle in the presence vs the absence of the light) was subsequently measured before and after 30, 60, or 90 presentations of the light without shock (extinction training). Thirty non-reinforced light presentations produced modest extinction, and 60 or 90 presentations produced nearly complete extinction (experiment 1). DCS injections (3.25, 15, or 30 mg/kg) before 30 non-reinforced light exposures dose-dependently enhanced extinction (experiment 2) but did not influence fear-potentiated startle in rats that did not receive extinction training (experiment 3). These effects were blocked by HA-966, an antagonist at the glycine-recognition site (experiment 4). Neither DCS nor HA-966 altered fear-potentiated startle when injected before testing (experiment 5). The effect of systemic administration was mimicked by intra-amygdala DCS (10 microg/side) infusions (experiment 6). These results indicate that treatments that promote NMDA receptor activity after either systemic or intra-amygdala administration promote the extinction of conditioned fear.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

The lysosome is a degradative organelle, and its fusion with other organelles is strictly regulated. In contrast to fusion with the late endosome, the mechanisms underlying autophagosome-lysosome fusion remain unknown. Here, we identify syntaxin 17 (Stx17) as the autophagosomal SNARE required for fusion with the endosome/lysosome. Stx17 localizes to the outer membrane of completed autophagosomes but not to the isolation membrane (unclosed intermediate structures); for this reason, the lysosome does not fuse with the isolation membrane. Stx17 interacts with SNAP-29 and the endosomal/lysosomal SNARE VAMP8. Depletion of Stx17 causes accumulation of autophagosomes without degradation. Stx17 has a unique C-terminal hairpin structure mediated by two tandem transmembrane domains containing glycine zipper-like motifs, which is essential for its association with the autophagosomal membrane. These findings reveal a mechanism by which the SNARE protein is available to the completed autophagosome.

Galectins, an ancient lectin family, are characterized by specific binding of beta-galactosides through evolutionary conserved sequence elements of carbohydrate-recognition domain (CRD). A structurally unique member of the family is galectin-3; in addition to the CRD it contains a proline- and glycine-rich N-terminal domain (ND) through which is able to form oligomers. Galectin-3 is widely spread among different types of cells and tissues, found intracellularly in nucleus and cytoplasm or secreted via non-classical pathway outside of cell, thus being found on the cell surface or in the extracellular space. Through specific interactions with a variety of intra- and extracellular proteins galectin-3 affects numerous biological processes and seems to be involved in different physiological and pathophysiological conditions, such as development, immune reactions, and neoplastic transformation and metastasis. The review attempts to summarize the existing information on structural, biochemical and intriguing functional properties of galectin-3.

Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.

Recent years have witnessed a steep increase in studies on the diverse roles of neuronal cation-chloride cotransporters (CCCs). The versatility of CCC gene transcription, posttranslational modification, and trafficking are on par with what is known about ion channels. The cell-specific and subcellular expression patterns of different CCC isoforms have a key role in modifying a neuron's electrophysiological phenotype during development, synaptic plasticity, and disease. While having a major role in controlling responses mediated by GABA(A) and glycine receptors, CCCs also show close interactions with glutamatergic signaling. A cross-talk among CCCs and trophic factors is important in short-term and long-term modification of neuronal properties. CCCs appear to be multifunctional proteins that are also involved in shaping neuronal structure at various stages of development, from stem cells to synaptogenesis. The rapidly expanding work on CCCs promotes our understanding of fundamental mechanisms that control brain development and functions under normal and pathophysiological conditions.

The proU locus encodes an osmotically inducible glycine betaine transport system that is important in the adaptation to osmotic stress. We present evidence that DNA supercoiling plays a key role in the osmotic induction of proU transcription. An increase in extracellular osmolarity increases in vivo DNA supercoiling, and the expression of proU is highly sensitive to these changes. Furthermore, topA mutations can mimic an increase in osmolarity, facilitating proU expression even in media of low osmolarity in which it is not normally expressed. Selection for trans-acting mutations that affect proU expression has yielded only mutations that alter DNA supercoiling, either in topA or a new genetic locus, osmZ, which strongly influences in vivo supercoiling. Mutations in osmZ are highly pleiotropic, affecting expression of a variety of chromosomal genes including ompF, ompC, fimA, and the bgl operon, as well as increasing the frequency of site-specific DNA inversions that mediate fimbrial phase variation.

Misfolding of the protein alpha-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved alpha-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix.

BACKGROUND

TDP-43 is a major component of the ubiquitinated inclusions that characterise amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitin inclusions (FTLD-U). TDP-43 is an RNA-binding and DNA-binding protein that has many functions and is encoded by the TAR DNA-binding protein gene (TARDBP) on chromosome 1. Our aim was to investigate whether TARDBP is a candidate disease gene for familial ALS that is not associated with mutations in superoxide dismutase 1 (SOD1).

METHODS

TARDBP was sequenced in 259 patients with ALS, FTLD, or both. We used TaqMan-based SNP genotyping to screen for the identified variants in control groups matched to two kindreds of patients for age and ethnic origin. Additional clinical, genetic, and pathological assessments were made in these two families.

RESULTS

We identified two variants in TARDBP, which would encode Gly290Ala and Gly298Ser forms of TDP-43, in two kindreds with familial ALS. The variants seem to be pathogenic because they co-segregated with disease in both families, were absent in controls, and were associated with TDP-43 neuropathology in both members of one of these families for whom CNS tissue was available.

CONCLUSIONS

The Gly290Ala and Gly298Ser mutations are located in the glycine-rich domain of TDP-43, which regulates gene expression and mediates protein-protein interactions such as those with heterogeneous ribonucleoproteins. Owing to the varied and important cellular functions of TDP-43, these mutations might cause neurodegeneration through both gains and losses of function. The finding of pathogenic mutations in TARDBP implicates TDP-43 as an active mediator of neurodegeneration in TDP-43 proteinopathies, a class of disorder that includes ALS and FTLD-U.

BACKGROUND

National Institutes of Health (AG10124, AG17586, AG005136-22, PO1 AG14382), Department of Veterans Affairs, Friedrich-Baur Stiftung (0017/2007), US Public Health Service, ALS Association, and Fundació 'la Caixa'.

Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA-amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography-mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.

The NMDA receptor is a key player in excitatory transmission and synaptic plasticity in the central nervous system. Its activation requires the binding of both glutamate and a co-agonist like D-serine to its glycine site. As D-serine is released exclusively by astrocytes, we studied the physiological impact of the glial environment on NMDA receptor-dependent activity and plasticity. To this end, we took advantage of the changing astrocytic ensheathing of neurons occurring in the supraoptic nucleus during lactation. We provide direct evidence that in this hypothalamic structure the endogenous co-agonist of NMDA receptors is D-serine and not glycine. Consequently, the degree of astrocytic coverage of neurons governs the level of glycine site occupancy on the NMDA receptor, thereby affecting their availability for activation and thus the activity dependence of long-term synaptic changes. Such a contribution of astrocytes to synaptic metaplasticity fuels the emerging concept that astrocytes are dynamic partners of brain signaling.

We report the in vivo targeting and imaging of tumor vasculature using arginine-glycine-aspartic acid (RGD) peptide-labeled quantum dots (QDs). Athymic nude mice bearing subcutaneous U87MG human glioblastoma tumors were administered QD705-RGD intravenously. The tumor fluorescence intensity reached maximum at 6 h postinjection with good contrast. The results reported here open up new perspectives for integrin-targeted near-infrared optical imaging and may aid in cancer detection and management including imaging-guided surgery.

Well-documented experimental evidence from both in vitro and in vivo models of stroke strongly supports the critical involvement of NMDA receptor-mediated excitotoxicity in neuronal damage after stroke. Despite this, the results of clinical trials testing NMDA receptor antagonists as neuroprotectants after stroke and brain trauma have been discouraging. Here, we report that in mature cortical cultures, activation of either synaptic or extrasynaptic NR2B-containing NMDA receptors results in excitotoxicity, increasing neuronal apoptosis. In contrast, activation of either synaptic or extrasynaptic NR2A-containing NMDA receptors promotes neuronal survival and exerts a neuroprotective action against both NMDA receptor-mediated and non-NMDA receptor-mediated neuronal damage. A similar opposing action of NR2B and NR2A in mediating cell death and cell survival was also observed in an in vivo rat model of focal ischemic stroke. Moreover, we found that blocking NR2B-mediated cell death was effective in reducing infarct volume only when the receptor antagonist was given before the onset of stroke and not 4.5 h after stroke. In great contrast, activation of NR2A-mediated cell survival signaling with administration of either glycine alone or in the presence of NR2B antagonist significantly attenuated ischemic brain damage even when delivered 4.5 h after stroke onset. Together, the present work provides a molecular basis for the dual roles of NMDA receptors in promoting neuronal survival and mediating neuronal damage and suggests that selective enhancement of NR2A-containing NMDA receptor activation with glycine may constitute a promising therapy for stroke.