Short-term synaptic enhancement and long-term potentiation in neocortex.

Repetitive stimuli reliably induce long-term potentiation (LTP) of synapses in the upper layers of the granular somatosensory cortex but not the agranular motor cortex of rats. Herein we examine, in these same cortical areas, short-term changes in synaptic strength that occur during the LTP induction period. theta-Burst stimulation produced a strong short-term enhancement of synapses in the granular area but only weak enhancement in the agranular area. The magnitude of enhancement during stimulation was strongly correlated with the magnitude of LTP subsequently expressed. Short-term enhancement was abolished by an antagonist of N-methyl-D-aspartate (NMDA) receptors but remained in the presence of a non-NMDA receptor antagonist. Inhibitory postsynaptic potentials of the granular and agranular areas displayed similar frequency sensitivity, but the frequency sensitivity of NMDA receptor-dependent excitatory postsynaptic potentials differed significantly between areas. We propose that pathway-specific differences in short-term enhancement are due to variations in the frequency dependence of NMDA currents; different capacities for short-term enhancement may explain why repetitive stimulation more readily induces LTP in the somatosensory cortex than in the motor cortex.

Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica.

Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 microM ZnCl2 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.

The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure.

Although the incidence of Gram-positive sepsis has risen strongly, it is unclear how Gram-positive organisms (without endotoxin) initiate septic shock. We investigated whether two cell wall components from Staphylococcus aureus, peptidoglycan (PepG) and lipoteichoic acid (LTA), can induce the inflammatory response and multiple organ dysfunction syndrome (MODS) associated with septic shock caused by Gram-positive organisms. In cultured macrophages, LTA (10 micrograms/ml), but not PepG (100 micrograms/ml), induces the release of nitric oxide measured as nitrite. PepG, however, caused a 4-fold increase in the production of nitrite elicited by LTA. Furthermore, PepG antibodies inhibited the release of nitrite elicited by killed S. aureus. Administration of both PepG (10 mg/kg; i.v.) and LTA (3 mg/kg; i.v.) in anesthetized rats resulted in the release of tumor necrosis factor alpha and interferon gamma and MODS, as indicated by a decrease in arterial oxygen pressure (lung) and an increase in plasma concentrations of bilirubin and alanine aminotransferase (liver), creatinine and urea (kidney), lipase (pancreas), and creatine kinase (heart or skeletal muscle). There was also the expression of inducible nitric oxide synthase in these organs, circulatory failure, and 50% mortality. These effects were not observed after administration of PepG or LTA alone. Even a high dose of LTA (10 mg/kg) causes only circulatory failure but no MODS. Thus, our results demonstrate that the two bacterial wall components, PepG and LTA, work together to cause systemic inflammation and multiple systems failure associated with Gram-positive organisms.

Synaptic activation of NF-kappa B by glutamate in cerebellar granule neurons in vitro.

Neuronal proliferation, migration, and differentiation are regulated by the sequential expression of particular genes at specific stages of development. Such processes rely on differential gene expression modulated through second-messenger systems. Early postnatal mouse cerebellar granule cells migrate into the internal granular layer and acquire differentiated properties. The neurotransmitter glutamate has been shown to play an important role in this developmental process. We show here by immunohistochemistry that the RelA subunit of the transcription factor NF-kappa B is present in several areas of the mouse brain. Moreover, immunofluorescence microscopy and electrophoretic mobility-shift assay demonstrate that in cerebellar granule cell cultures derived from 3- to 7-day-old mice, glutamate specifically activates the transcription factor NF-kappa B, as shown by binding of nuclear extract proteins to a synthetic oligonucleotide reproducing the kappa B site of human immunodeficiency virus. The use of different antagonists of the glutamate recpetors indicates that the effect of glutamate occurs mainly via N-methyl-D-aspartate (NMDA)-receptor activation, possibly as a result of an increase in intracellular Ca2+. The synaptic specificity of the effect is strongly suggested by the observation that glutamate failed to activate NF-kappa B in astrocytes, while cytokines, such as interleukin 1 alpha and tumor necrosis factor alpha, did so. The effect of glutamate appears to be developmentally regulated. Indeed, NF-kappa B is found in an inducible form in the cytoplasm of neurons of 3- to 7-day-old mice but is constitutively activated in the nuclei of neurons derived from older pups (8-10 days postnatal). Overall, these observations suggest the existence of a new pathway of trans-synaptic regulation of gene expression.

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.

Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors.

Stimulation of muscarinic m1 or m3 receptors can, by generating diacylglycerol and activating protein kinase C, accelerate the breakdown of the amyloid precursor protein (APP) to form soluble, nonamyloidogenic derivatives (APPs), as previously shown. This relationship has been demonstrated in human glioma and neuroblastoma cells, as well as in transfected human embryonic kidney 293 cells and PC-12 cells. We now provide evidence that stimulation of metabotropic glutamate receptors (mGluRs), which also are coupled to phosphatidylinositol 4,5-bisphosphate hydrolysis, similarly accelerates processing of APP into nonamyloidogenic APPs. This process is demonstrated both in hippocampal neurons derived from fetal rats and in human embryonic kidney 293 cells transfected with cDNA expression constructs encoding the mGluR 1 alpha subtype. In hippocampal neurons, both an mGluR antagonist, L-(+)-2-amino-3-phosphonopropionic acid, and an inhibitor of protein kinase C, GF 109203X, blocked the APPs release evoked by glutamate receptor stimulation. Ionotropic glutamate agonists, N-methyl-D-aspartate or S(-)-5-fluorowillardiine, failed to affect APPs release. These data show that selective mGluR agonists that initiate signal-transduction events can regulate APP processing in bona fide primary neurons and transfected cells. As glutamatergic neurons in the cortex and hippocampus are damaged in Alzheimer disease, amyloid production in these regions may be enhanced by deficits in glutamatergic neurotransmission.

Tissue specificity of a glucocorticoid-dependent enhancer in transgenic mice.

The glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase were associated with the regulatory sequences of a cellular gene expressed ubiquitously--that coding for the largest subunit of RNA polymerase II. In transient expression assays, glucocorticoid responsiveness of the hybrid regulatory regions depends on the spatial relationship and number of regulatory elements. Two parameters affect the ratio of induction by glucocorticoids: the basal level of the hybrid promoter that is affected by the RNA polymerase II regulatory sequences and the glucocorticoid-induced level that depends on the distance between the GRUs and the TATA box. A fully active glucocorticoid-responsive hybrid gene was used to generate transgenic mice. Results show that a composite regulatory pattern is obtained: ubiquitous basal expression characteristic of the RNA polymerase II gene and liver-specific glucocorticoid activation characteristic of the tyrosine aminotransferase GRUs. This result demonstrates that the activity of the tyrosine aminotransferase GRUs is cell-type-specific not only in cultured cells but also in the whole animal.

Two distinct oscillators in the rat suprachiasmatic nucleus in vitro.

In the rat suprachiasmatic nucleus slice culture, circadian rhythms in the release of arginine vasopressin and vasoactive intestinal polypeptide were measured simultaneously and longitudinally. The phase relationship between the two peptide rhythms was relatively constant in the culture without a treatment of antimitotic drugs but became diverse by an introduction of antimitotics, which is generally used to reduce the number of glial cells. By monitoring the two rhythms continuously for 6 days, different periods were detected in culture with the antimitotic treatment. Furthermore, N-methyl-D-aspartate shifted the phase of the two peptide rhythms in the same culture differently. These results indicate that the arginine vasopressin and vasoactive intestinal polypeptide release are under control of different circadian oscillators.

Somatodendritic expression of an immediate early gene is regulated by synaptic activity.

Trans-synaptic activation of gene expression is linked to long-term plastic adaptations in the nervous system. To examine the molecular program induced by synaptic activity, we have employed molecular cloning techniques to identify an immediate early gene that is rapidly induced in the brain. We here report the entire nucleotide sequence of the cDNA, which encodes an open reading frame of 396 amino acids. Within the hippocampus, constitutive expression was low. Basal levels of expression in the cortex were high but can be markedly reduced by blockade of N-methyl-D-aspartate receptors. By contrast, synaptic activity induced by convulsive seizures increased mRNA levels in neurons of the cortex and hippocampus. High-frequency stimulation of the perforant path resulted in long-term potentiation and a spatially confined dramatic increase in the level of mRNA in the granule cells of the ipsilateral dentate gyrus. Transcripts were localized to the soma and to the dendrites of the granule cells. The dendritic localization of the transcripts offers the potential for local synthesis of the protein at activated postsynaptic sites and may underlie synapse-specific modifications during long-term plastic events.

Further studies of the role of Ser-16 in the regulation of the activity of phenylalanine hydroxylase.

It was previously proposed that the activation of rat liver phenylalanine hydroxylase (EC 1.14.16.1) by cAMP-dependent protein kinase-mediated phosphorylation of Ser-16 is due to the introduction of the negatively charged phosphate group. To explore the validity of this proposal, we have applied site-directed mutagenesis to specifically replace Ser-16 with negatively charged amino acids, glutamic and aspartic; with polar uncharged amino acids, asparagine and glutamine; with the positively charged amino acid lysine; and with the nonpolar hydrophobic amino acid alanine. The wild-type and mutant enzymes were purified to homogeneity, and the importance of Ser-16 in the activation of phenylalanine hydroxylase was examined by comparing the state of activation of the phosphorylated form of the wild-type hydroxylase with that of the mutants. The kinetic studies carried out on the wild-type phosphorylated hydroxylase showed that all the activation could be accounted for by an increase in Vmax with no change in Km for either phenylalanine or the pterin cofactor. Replacement of Ser-16 with a negatively charged residue, glutamate of aspartate, resulted in the activation of the hydroxylase by 2- to 4-fold, whereas replacement with glutamine, asparagine, lysine, or alanine resulted in a much more modest increase. Further, lysolecithin was found to stimulate the phosphorylated hydroxylase and the mutant enzymes S16E and S16D by a factor of 6-7. In contrast, the mutants S16Q, S16N, and S16A all showed the same magnitude of activation as the wild-type with lysolecithin. Therefore, this study demonstrates that activation of the enzyme by phosphorylation of Ser-16 by cAMP-dependent protein kinase is due to the introduction of negative charge(s) and strongly suggests the involvement of electrostatic interaction between the regulatory and catalytic domains of the hydroxylase.

Structural conservation of ion conduction pathways in K channels and glutamate receptors.

Single channel recordings demonstrate that ion channels switch stochastically between an open and a closed pore conformation. In search of a structural explanation for this universal open/close behavior, we have uncovered a striking degree of amino acid homology across the pore-forming regions of voltage-gated K channels and glutamate receptors. This suggested that the pores of these otherwise unrelated classes of channels could be structurally conserved. Strong experimental evidence supports a hairpin structure for the pore-forming region of K channels. Consequently, we hypothesized the existence of a similar structure for the pore of glutamate receptors. In ligand-gated channels, the pore is formed by M2, the second of four putative transmembrane segments. A hairpin structure for M2 would affect the subsequent membrane topology, inverting the proposed orientation of the next segments, M3. We have tested this idea for the NR1 subunit of the N-methyl-D-aspartate receptor. Mutations that affected the glycosylation pattern of the NR1 subunit localize both extremes of the M3-M4 linker to the extracellular space. Whole cell currents and apparent agonist affinities were not affected by these mutations. Therefore it can be assumed that they represent the native transmembrane topology. The extracellular assignment of the M3-M4 linker challenged the current topology model by inverting M3. Taken together, the amino acid homology and the new topology suggest that the pore-forming M2 segment of glutamate receptors does not transverse the membrane but, rather, forms a hairpin structure, similar to that found in K channels.

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.

Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.

D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.

Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.