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At least 1,063 records · Page 59Linked to original sources
PubMed1997

Arginine-glycine-aspartic acid mimics can identify a transitional activation state of recombinant alphaIIb beta3 in human embryonic kidney 293 cells.

The platelet-specific integrin alphaIIb beta3 achieves a high affinity binding state in response to extracellular agonists such as thrombin, ADP, or collagen. During this activation, the receptor undergoes a number of conformational changes. To characterize the different conformations of alphaIIb beta3, we expressed recombinant alphaIIb beta3 in human embryonic kidney (HEK) 293 cells. Antigenic and peptide recognition specificities of the full-length recombinant receptor resembled those of the native receptor in platelets. We used an array of peptidic and nonpeptidic arginine-glycine-aspartic acid (RGD) mimics that specifically bind to human platelet alphaIIb beta3 to determine the affinity state of the receptor. Some of these RGD mimics were previously shown to clearly discriminate between resting and activated alphaIIb beta3. Solution-phase binding of these RGD mimics to the recombinant cells suggested that in HEK 293 cells the full-length alphaIIb beta3 is expressed in a "transitional" activation state. This observation was confirmed by the binding of the activation-specific, monoclonal anti-alphaIIb beta3 antibody PAC1 to cells expressing the full-length recombinant alphaIIb beta3. Deletion of the entire cytoplasmic domain of the beta subunit was sufficient to convert the receptor in HEK 293 cells to a fully active form, as found in activated platelets. In addition, the full-length receptor was capable of mediating agonist-independent aggregation of cells in the presence of fibrinogen. Thus, by using RGD mimics, we have identified a functional transitional activation state of alphaIIb beta3 that is capable of mediating fibrinogen-dependent cell aggregation.

Antigens, CD↗
PubMed2002

Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen.

Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen.

Amino Acid Sequence↗
PubMed2003

Morphine-3-glucuronide's neuro-excitatory effects are mediated via indirect activation of N-methyl-D-aspartic acid receptors: mechanistic studies in embryonic cultured hippocampal neurones.

UNLABELLED: Indirect evidence indicates that morphine-3-glucuronide (M3G) may contribute significantly to the neuro-excitatory side effects (myoclonus and allodynia) of large-dose systemic morphine. To gain insight into the mechanism underlying M3G's excitatory behaviors, we used fluo-3 fluorescence digital imaging techniques to assess the acute effects of M3G (5-500 microM) on the cytosolic calcium concentration ([Ca(2+)](CYT)) in cultured embryonic hippocampal neurones. Acute (3 min) exposure of neurones to M3G evoked [Ca(2+)](CYT) transients that were typically either (a) transient oscillatory responses characterized by a rapid increase in [Ca(2+)](CYT) oscillation amplitude that was sustained for at least approximately 30 s or (b) a sustained increase in [Ca(2+)](CYT) that slowly recovered to baseline. Naloxone-pretreatment decreased the proportion of M3G-responsive neurones by 10%-25%, implicating a predominantly non-opioidergic mechanism. Although the naloxone-insensitive M3G-induced increases in [Ca(2+)](CYT) were completely blocked by N-methyl-D-aspartic acid (NMDA) antagonists and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate antagonist), CNQX did not block the large increase in [Ca(2+)](CYT) evoked by NMDA (as expected), confirming that M3G indirectly activates the NMDA receptor. Additionally, tetrodotoxin (Na(+) channel blocker), baclofen (gamma-aminobutyric acid(B) agonist), MVIIC (P/Q-type calcium channel blocker), and nifedipine (L-type calcium channel blocker) all abolished M3G-induced increases in [Ca(2+)](CYT), suggesting that M3G may produce its neuro-excitatory effects by modulating neurotransmitter release. However, additional characterization is required. IMPLICATIONS: Large systemic doses of morphine administered to some patients for cancer pain management have been reported to produce myoclonus and allodynia. Indirect evidence implicates the major morphine metabolite, morphine-3-glucuronide (M3G), in these neuro-excitatory side effects. Hence, this study was designed to gain insight into the cellular mechanism responsible for M3G's neuro-excitatory actions.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1993

Site-directed mutagenesis of the arginine-glycine-aspartic acid in vitronectin abolishes cell adhesion.

Vitronectin (VN), a major cell adhesion protein, is found in plasma and in the extracellular matrix. At least three distinct cell surface receptors for vitronectin belonging to the integrin superfamily have been identified in normal and neoplastic cells. Many cell adhesion ligands, including vitronectin, contain an Arg-Gly-Asp (RGD) sequence mediating, in part, the ligand-receptor interaction. These ligands bind different integrins with varying specificity and affinity. The mechanism of receptor specificity remains controversial. To determine the role of the RGD sequence in receptor specificity, we amplified the cDNA for human vitronectin from a liver cDNA library and generated two separate mutants by utilizing site-directed mutagenesis resulting in aspartic acid (Asp47) to glutamic acid (Glu47) substitution and glycine (Gly46) to alanine (Ala46) substitution. The mammalian expression vector, pZEM229R, was used to transfect baby hamster kidney cells which secreted recombinant proteins into the supernatant. All recombinant proteins were isolated by heparin-agarose chromatography and tested for interaction with three known vitronectin receptors, namely, alpha IIIb beta 3 on thrombin-activated platelets, alpha v beta 3 on human umbilical vein endothelial cells and alpha v beta 5 on Panc-1 cells. Recombinant wild-type vitronectin behaved in a fashion similar to plasma-derived vitronectin. Both the RGE-VN and RAD-VN recombinant mutant proteins showed complete loss of cell adhesion activity, regardless of the receptor. These results confirm the essential and central role of the RGD sequence in vitronectin for cell adhesion. This expression system allows further structure/function analysis of vitronectin.

Amino Acid Sequence↗
PubMed2005

Site-specific phosphorylation of SCG10 in neuronal plasticity: role of Ser73 phosphorylation by N-methyl D-aspartic acid receptor activation in rat hippocampus.

Accumulated evidence suggests that actin and microtubule regulating proteins contribute to neuronal structural dynamics, which subsequently affect neuronal plasticity. SCG10 is a neuronal-specific stathmin protein with microtubule destabilizing activity that is affected by multiple phosphorylation, at least in vitro. SCG10 has four major phosphorylation sites: Ser50 and Ser97 targeted by protein kinase A (PKA), and Ser62 and Ser73 targeted by mitogen-activated protein kinase (MAPK). To explore the potential roles of site-specific phosphorylation in physiological models, we developed phosphorylation site-specific antibodies and examined the SCG10 status in primary cultured hippocampal neurons and tissues. Although SCG10 is concentrated in growth cones and the Golgi apparatus in primary cultured neurons, the phosphorylated form was also detected in both regions, suggesting that MT dynamics within the growth cone may be regulated by protein phosphorylation. In the adult hippocampus, an intense stimulus such as kainate treatment induced a rapid phosphorylation of Ser73 within 15 min that was sustained for at least 60 min. This response was mediated through the N-methyl D-aspartic acid (NMDA) receptor and was ablated by the antagonist MK-801. The MAPK enzyme Erk2 was simultaneously activated along a similar time course to SCG10, suggesting that Erk2 may directly phosphorylate Ser73. These results demonstrate that changes in the phosphorylation status of SCG10 in vivo, dependent upon neural activity and/or plasticity, could affect the microtubule dynamics in neuronal dendrites.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of properdin deficiency type III: dysfunction produced by a single point mutation in exon 9 of the structural gene causing a tyrosine to aspartic acid interchange.

Inherited properdin deficiency is an X-linked recessive disorder clinically manifested by susceptibility to meningococcal disease. Deficiency of properdin is characterized by complete absence (type I), very low level presence (type II), or the presence of a dysfunctional properdin protein in serum as found in one Dutch family (type III). To better understand the dysfunctional protein on the molecular level, samples from three members of the Dutch family were analyzed by direct genomic sequencing. The sequence of the complete gene, including 10 exons and 9 introns, covering about 6500 bases was determined. The dysfunctional properdin was found to be caused by a single T to G mutation in exon 9, which gives rise to a substitution of a tyrosine by an aspartic acid residue at position 387. This change to a hydrophilic amino acid affects the function of the properdin molecule, although the oligomerization of dysfunctional properdin molecules was similar to that of normal properdin. In binding studies with C3b and properdin in serum, no properdin deposition was detected with the type III deficient serum. Inhibition studies with different decapeptides revealed distinct inhibitory sequences, and indicated also that the part of properdin containing the type III mutation was not directly involved in the binding to C3b. The mutation most likely causes conformational changes that make the properdin molecule dysfunctional by affecting its binding to C3b.

Amino Acid Sequence↗
PubMed1998

Strychnine- and bicuculline-induced changes in the firing pattern of motoneurones during in vitro fictive locomotion reveal a possible N-methyl-D-aspartic acid (NMDA)-mediated suppression of motor discharge in wallaby (Macropus eugenii) pouch young.

Inhibitory pathways in the spinal cord play an important role in establishing the pattern of motor discharge. In the wallaby spinal cord preparation, disruption of glycinergic and gamma-amino butyric acid (GABA)ergic neurotransmission abolished the alternation between antagonistic motor pools during fictive locomotion. A new pattern of motor discharge also appeared when both glycine and GABA(A) receptors were blocked simultaneously. This discharge pattern was biphasic, characterized by a distinct pause between two bursts of motoneurone firing during each cycle of motor activity. Whole cell patch recordings showed that the second burst of motor discharge was not caused by a separate inward current at a delayed time course. Furthermore, local injection of an N-methyl-D-aspartic acid (NMDA) specific antagonist converted the biphasic discharge to a continuous burst pattern. The result suggests an NMDA-mediated mechanism, which causes a suppression of motoneurone firing when glutamate release from interneurones is enhanced in the absence of glycinergic and GABAergic inhibition.

Animals↗
PubMed2003

Transforming growth factor-alpha attenuates N-methyl-D-aspartic acid toxicity in cortical cultures by preventing protein synthesis inhibition through an Erk1/2-dependent mechanism.

Transforming growth factor-alpha (TGF-alpha), a ligand of the epidermal growth factor receptor, reduces the infarct size after focal cerebral ischemia in rat, but the molecular basis underlying the protection is unknown. Excitotoxicity and global inhibition of translation are acknowledged to contribute significantly to the ischemic damage. Here we studied whether TGF-alpha can rescue neurons from excitotoxicity in vitro and how it affects calcium homeostasis, protein synthesis, and the associated Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) intracellular signaling pathways in mixed neuron-glia cortical cultures. We found that 100 ng/ml TGF-alpha attenuated neuronal cell death induced by a 30-min exposure to 35 microM N-methyl-D-aspartic acid (NMDA) (as it reduced lactate dehydrogenase release, propidium iodide staining, and caspase-3 activation) and decreased the elevation of intracellular Ca2+ elicited by NMDA. TGF-alpha induced a prompt and sustained phosphorylation of Erk1/2 and prevented the loss of Akt-P induced by NMDA 3 h after exposure. The protective effect of TGF-alpha was completely prevented by PD 98059, an inhibitor of the Erk1/2 pathway. Studies of incorporation of [3H]leucine into proteins showed that NMDA decreased the rate of protein synthesis, and TGF-alpha attenuated this effect. TGF-alpha stimulated the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) but did not affect eIF2 alpha, two proteins involved in translation regulation. PD 98059 abrogated the TGF-alpha effect on eIF4E. Our data demonstrate that TGF-alpha exerts a neuroprotective action against NMDA toxicity, in which Erk1/2 activation plays a key role, and suggest that the underlying mechanisms involve recovery of translation inhibition, mediated at least in part by eIF4E phosphorylation.

Animals↗
PubMed1995

Luteinizing hormone response to N-methyl-D, L-aspartic acid in the presence of physiological estradiol concentrations: influence of age and the ovary.

We have previously reported that the pituitary of intra-atrially cannulated old female C57BL/6J mice is as capable of responding to a GnRH challenge as is that of young females (10). We have observed elevated luteinizing hormone (LH) levels in ovariectomized (OVX) intra-atrially cannulated mice. Sustained physiologic levels of estradiol (E2) for 6 days suppressed circulating LH to intact levels. However, in that model, a bolus of E2 following E2 priming was unable to elicit an LH surge (Joshi et al., unpublished findings). The present studies were designed to examine: first, whether GnRH neurons are competent to release GnRH in the presence of tonic physiologic levels of E2 and, second, whether either age or the ovary can influence GnRH neuronal responsiveness. The N-methyl-D, L-aspartic acid (NMA)-evoked GnRH response was assessed indirectly by measuring LH in two groups of OVX C57BL/6J mice: short-term OVX (S-OVX) (1 week) mice were either prepubertal (5 weeks), postpubertal (10 weeks), young (5 months), middle aged (12 months), or old (24 months). Long-term OVX (L-OVX) mice were either young (5 months), or old (24 months) and OVX at puberty; middle-aged L-OVX mice were OVX at 8 months and examined at 12 months of age. Animals were administered physiologic levels of E2 by subcutaneous silastic capsule for 1 week before testing. LH secretion was inhibited by E2 in S-OVX mice of all ages. In no case did NMA overcome this inhibition in E2 primed S-OVX females. E2 also inhibited LH secretion in L-OVX mice of all ages, but NMA was able to overcome the E2 inhibition of LH secretion in L-OVX mice (young: 0.5 +/- 0.1, 0.84 +/- 0.19 ng/ml, first and second challenge, respectively; middle-aged: 0.46 +/- 0.1, 1.08 +/- 0.16 ng/ml; and old: 1.44 +/- 0.19, 0.99 +/- 0.27 ng/ml). This last effect was independent of animal maturity at the time of OVX or animal age at the time of experiment. These findings suggest that although the ovaries in the 24-month-old S-OVX mice had not produced enough E2 to alter the vaginal cytology for 2 +/- 0.5 months before the experiment, the ovarian modulation of the inhibitory effect of E2 on NMA-induced LH secretion was still present. The nature of the ovarian factor(s) modulating this effect is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Age Factors↗
PubMed1995

Long-term potentiation of C-fiber-evoked potentials in the rat spinal dorsal horn is prevented by spinal N-methyl-D-aspartic acid receptor blockage.

Long-term potentiation (LTP) of synaptic potentials is a fundamental mechanism of memory formation in the hippocampus. Here, we have characterized long-term changes of field potentials which were evoked in the lumbar spinal dorsal horn by supramaximal electrical stimulation of the sciatic nerve in urethane anesthetized rats. The field potentials had high thresholds (> or = 7 V), long latencies (90-130 ms, corresponding to conduction velocities between 1.2 and 0.85 m/s) and were not affected by spinalization (at C5-C6) or muscle relaxation (with pancuronium), i.e. the potentials were probably evoked by afferent C-fibers. Tetanic electrical stimulation (0.5 ms pulses, 30-40 V, 100 Hz, given in 4 trains of 1 s duration at 10 s intervals) of sciatic nerve induced in all 9 rats tested a LTP of amplitude of the C-fiber-evoked potential throughout recording periods which lasted between 4 and 9 h. Mean potentiation ranged from +71% to +174%. Superfusion of spinal cord with N-methyl-D-aspartic acid (NMDA) receptor antagonist D-(-)-4-(3-phosphonopropyl)piperazine-2-carboxylic (500 nM), which has little effect on the amplitude of C-fiber-evoked potentials, completely blocked LTP induced by tetanic stimulation in all five rats tested. Superfusion of spinal cord with NMDA (1 microM, 10 microM or 50 microM) induced LTP in only 2 out of 8 rats. This is the first report showing that LTP of C-fiber-evoked field potentials in the spinal dorsal horn in vivo may last for more than 8 h. This LTP in the spinal dorsal horn may underlie plastic changes of spinal nociception.

Animals↗
PubMed1994

Relief of post-herpetic neuralgia with the N-methyl-D-aspartic acid receptor antagonist ketamine: a double-blind, cross-over comparison with morphine and placebo.

Pain and sensory thresholds were examined before and after intravenous administration of ketamine (0.15 mg/kg), morphine (0.075 mg/kg) or saline in 8 patients with post-herpetic neuralgia. A randomized, double-blind, cross-over study design was used. Post-herpetic neuralgia was associated with impaired sensory function, as shown by reduced tactile and warm sensation in the affected compared with the contralateral non-affected skin area. Neither ketamine nor morphine changed significantly the thresholds for warm, cold, heat pain or tactile sensation. However, ketamine normalized abnormal heat pain sensations in 4 patients, probably due to a central effect. Ketamine, but not morphine, produced significant relief of pain. Pain evoked by non-noxious stimulation of the skin (allodynia) was significantly inhibited by ketamine as well as by morphine. Wind-up-like pain (i.e., pain evoked by repeatedly pricking the affected skin area) was significantly inhibited by ketamine, but significantly aggravated by morphine. Side effects were observed in all the 8 patients after injection of ketamine and in 6 patients after injection of morphine. The present results support the hypothesis that the N-methyl-D-aspartic acid (NMDA) receptors are involved in the control of post-herpetic neuralgia including allodynia and wind-up-like pain. The NMDA receptors also may play a role in the modulation of thermal perception.

Aged↗