Biomedical subjects

J Spiess

Publications and source records attributed to J Spiess.

At least 55 records · Page 3Linked to original sources

PubMed1998

Mapping of the ligand-selective domain of the Xenopus laevis corticotropin-releasing factor receptor 1: implications for the ligand-binding site.

The nonselective human corticotropin-releasing factor receptor 1 (hCRF-R1) and the ligand-selective Xenopus CRF-R1 (xCRF-R1) were compared. To understand the interactions of sauvagine and ovine CRF, both high-affinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF-R1 followed by double or multiple point mutations were constructed. Binding studies and cAMP assays demonstrated that the N-terminal domain exhibited the complete ligand selectivity of xCRF-R1. The important region was mapped between amino acids 70 and 89; replacement of amino acids Arg76, Asn81, Gly83, Leu88, and Ala89 in hCRF-R1 with the corresponding amino acids of xCRF-R1 (Gln76, Gly81, Val83, His88, and Leu89) resulted in a receptor that had approximately 30-fold higher affinity for human/rat CRF than for sauvagine. Mutagenesis of these amino acids in xCRF-R1 to the human sequence completely abolished the ligand selectivity of xCRF-R1. Mutagenesis of amino acids 88 and 89 in hCRF-R1 or xCRF-R1 had only a minor (approximately 2.5-fold) effect on the ligand selectivity of the mutant receptor. Substitution of Arg76, Asn81, and Gly83 in hCRF-R1 with the corresponding sequence of xCRF-R1 (Gln76, Gly81, and Val83) resulted in a receptor with approximately 11-fold higher affinity for human/rat CRF compared with ovine CRF or sauvagine. When only two of these three amino acids were mutated, no effect on the ligand selectivity was observed. On the basis of these data, it is suggested that amino acids 70-89 of CRF-R1 are important for the ligand binding site.

Amino Acid Sequence↗

PubMed1998

Production of the Fos protein after contextual fear conditioning of C57BL/6N mice.

Male C57BL/6N mice were chosen to determine Fos production during acquisition of context-dependent fear and after re-exposure to the conditioning context. Fear-conditioning was induced by a single exposure of mice to a context followed by an electric shock. Control groups consisted of mice exposed to context only (Context group) or to an immediate electric shock. When contextual retention was measured 24 h after conditioning (retention test 1), significant contextual generalization was observed. However, when animals were exposed to a different context from days 2-5 after conditioning and then tested for retention on day 6 (retention test 2), generalization was markedly reduced. After the training, the fear-conditioned mice produced higher Fos levels than mice exposed to an immediate shock in the hippocampus, medial amygdaloid nucleus and parietal somatosensory cortex. Both shock groups produced significantly more Fos than the Context group in the central nucleus of the amygdala. After retention test 1, fear-conditioned mice generated more Fos in the hippocampus and central amygdaloid nucleus than the two control groups. However, all groups exhibited similarly low Fos production after retention test 2. The results demonstrated that simultaneous Fos production in the hippocampus, central and medial nuclei of amygdala and somatosensory parietal cortex closely paralleled the ability of mice to acquire conditioned fear. In contrast, Fos production after the retention tests did not correlate with the expression of conditioned fear.

Amygdala↗

PubMed1998

Generalization of fear responses in C57BL/6N mice subjected to one-trial foreground contextual fear conditioning.

C57BL/6N mice were trained in a foreground contextual fear conditioning paradigm by a single exposure to a context (context 1) paired with an electric shock. Conditioned mice exhibited a strong fear response, indicated by increased freezing and low locomotor activity after subsequent re-exposure to context 1, which had been used for conditioning as well as in a novel context (context 2). The fear responses to contexts 1 or 2 required the temporal sequence of the conditioned (CS) and unconditioned (US) stimuli and did not differ significantly. The intensity of the fear response was maximal 24 h after conditioning, when long-term memory was fully established. The response to context 2 was interpreted as conditioned generalization, which was also observed in the elevated plus-maze representing an environment which differed in its spatial design significantly from contexts 1 and 2. Contextual discrimination of C57BL/6N mice was enhanced after extinction of the generalized fear. Strain studies employing C57BL/6J and Balb/c mice in addition to the C57BL/6N strain revealed that Balb/c and C57BL/6J mice acquired significantly weaker conditioned fear and generalized significantly less than C57BL/6N mice. It was concluded that the intensities of the context-specific and generalized fear responses were interlinked in a strain-specific manner.

Animals↗

PubMed1998

A synthetic glucagon-like peptide-1 analog with improved plasma stability.

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

Animals↗

PubMed1997

The phosphoprotein DARPP-32 mediates cAMP-dependent potentiation of striatal N-methyl-D-aspartate responses.

The signal transduction pathway underlying the cAMP-dependent modulation of rat striatal N-methyl-D-aspartate (NMDA) responses was investigated by using the two-electrode voltage-clamp technique. In oocytes injected with rat striatal poly(A)+ mRNA, activation of cAMP-dependent protein kinase (PKA) by forskolin potentiated NMDA responses. Inhibition of protein phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A) by the specific inhibitor calyculin A occluded the PKA-mediated potentiation of striatal NMDA responses, suggesting that the PKA effect was mediated by inhibition of a protein phosphatase. Coinjection of oocytes with striatal mRNA and antisense oligodeoxynucleotides directed against the protein phosphatase inhibitor DARPP-32 dramatically reduced the PKA enhancement of NMDA responses. NMDA responses recorded from oocytes injected with rat hippocampal poly(A)+ mRNA were not affected by stimulation of PKA. When oocytes were coinjected with rat hippocampal poly(A)+ mRNA plus complementary RNA coding for DARPP-32, NMDA responses were potentiated after stimulation of PKA. The results provide evidence that DARPP-32, which is enriched in the striatum, may participate in the signaling between the two major afferent striatal pathways, the glutamatergic and the dopaminergic projections, by the cAMP-dependent regulation of striatal NMDA currents.

Animals↗

PubMed1997

Structure-function relationship of different domains of the rat corticotropin-releasing factor receptor.

The significance of different domains of corticotropin-releasing factor receptor, type 1, (CRFR1) for ligand binding and cAMP accumulation was investigated with C-terminally truncated forms of rat CRFR1 (rCRFR1) tagged by a sequence of six histidine residues (His-tag) to facilitate protein purification and identification. These different forms of the receptor were N-glycosylated and transported properly to the membranes of transfected mammalian cells as indicated by Western blot analysis and immunocytochemical staining with two polyclonal antibodies developed against the N- and C-terminus of rCRFR1. The N-terminal fragment, rCRFR1(23-121), expressed in Escherichia coli bound oCRF specifically, but with low affinity. Several mutants lacking transmembrane domain (TM) 7 and the C-terminus exhibited similarly low affinities to oCRF after expression in transfected mammalian cells. None of these cells produced significant amounts of cAMP after exposure to oCRF. Only mutants containing the N-terminus, all loops and TMs bound oCRF and produced cAMP with high affinity (Kd = 62 nM) and efficacy (EC50 = 0.8 nM). The additional presence of the C-terminus provided similar characteristics (Kd = 5 nM, EC50 = 0.3 nM) as known for the native receptor. It is suggested on the basis of these data that the last extracellular loop is involved in ligand binding.

Amino Acid Sequence↗

PubMed1997

Modulation of the inflammatory response by corticotropin-releasing factor.

Peptides of the corticotropin-releasing factor (CRF) family have been shown to have either pro- or anti-inflammatory activities. CRF (10-30 micrograms/kg) administered subcutaneously or intravenously could inhibit edema and dye leakage in the rat paw produced by several injuries. These findings are opposed to some results suggesting a predominantly pro-inflammatory effect of CRF mainly in arthritic processes. The purpose of this work was to identify in vivo and in vitro the conditions for the pro- or anti-inflammatory actions of CRF in order to clarify its physiological and pharmacological function. Using the rat paw edema test we observed that only the highest doses of CRF employed (5 micrograms) induced a moderate and sustained swelling. Pre-treatment with low doses of CRF (0.5-5 ng) was able to inhibit the edema induced by Naja naja phospholipase A2, carrageenin or histamine. Higher doses (50 ng-5 micrograms) had no anti-inflammatory activity. When co-inhibited with Naja naja phospholipase A2 or histamine the peptide did not modify the swelling at doses up to 500 ng, showing at 5 micrograms an additive edema with Naja naja phospholipase A2. In vitro, CRF did not modify the release of histamine but slightly increased the release of arachidonic acid to the medium. Our findings show a clear dose dependence on the local effects of CRF in inflammatory responses. These results suggest that the mechanisms of the two dose-related phenomena may be distinct.

Animals↗

PubMed1997

Effect of tone-dependent fear conditioning on heart rate and behavior of C57BL/6N mice.

The effects of the temporal sequence of tone (conditioned stimulus [CS]) and footshock (unconditioned stimulus [US]) during training (acquisition) on tone-dependent retention were studied in mice. Freezing increased significantly as an associative behavioral response in mice subjected to CS paired with US or after unpaired by 30 s in the memory test performed 24 hr after training. In the home cage of freely moving mice implanted with an electrocardiogram transmitter, CS triggered a strong tachycardiac response in the memory test. Heart rate (HR) increased from about 580 bpm to more than 750 bpm, and HR variability decreased significantly. The inhibition of the HR increase by the nonspecific beta-adrenergic antagonist sotalol indicated the strong sympathetic contribution to the tachycardiac response. CS evoked a significant but minor HR increase in mice subjected to either CS or US only or CS and US unpaired by 60 s. Thus, HR and HR variability reflected associative learning.

Animals↗

PubMed1997

Regulation of corticotropin-releasing factor receptor function in human Y-79 retinoblastoma cells: rapid and reversible homologous desensitization but prolonged recovery.

Homologous receptor desensitization is an important regulatory response to continuous activation by agonist that involves the uncoupling of a receptor from its G protein. When human retinoblastoma Y-79 cells expressing corticotropin-releasing factor (CRF) receptors were preincubated with CRF for 10 min-4 h, a time-dependent reduction in both the peak and sensitivity of CRF-stimulated intracellular cyclic AMP (cAMP) accumulation developed with a t1/2 of 38 min and an EC50 of 6-7 nM CRF. CRF receptor desensitization was slowly reversible after a 4-h CRF preincubation with a t1/2 of 13 h and a full restoration of cAMP responsiveness to CRF at 24 h following the removal of 10 nM CRF. Because the ability of vasoactive intestinal peptide, forskolin, or (-)-isoproterenol to stimulate cAMP accumulation was not diminished in Y-79 cells desensitized with 10 nM CRF, the observed desensitization was considered to be a specific homologous action of CRF. CRF receptor desensitization was markedly attenuated by CRF receptor antagonists, which alone did not produce any appreciable reduction in CRF-stimulated cAMP accumulation. Although recent reports have demonstrated a rapid decline in steady-state levels of CRF receptor type 1 (CRF-R1) mRNA in anterior pituitary cells during several hours of exposure to CRF, there was no observed reduction in CRF-R1 mRNA levels when Y-79 cells were preincubated with 10 nM CRF for 10 min-24 h despite a rapid time- and concentration-dependent loss of CRF receptors from the retinoblastoma cell surface.

Adenylyl Cyclases↗

PubMed1997

Identification of two corticotropin-releasing factor receptors from Xenopus laevis with high ligand selectivity: unusual pharmacology of the type 1 receptor.

Two cDNA clones encoding distinct members of the corticotropin-releasing factor (CRF) receptor family have been isolated from Xenopus laevis with PCR-based approaches. The first full-length cDNA amplified from Xenopus brain encoded a 415-amino acid protein with approximately 80% identity to mammalian CRF receptor type 1 (CRF-R1). The second full-length cDNA isolated from Xenopus brain and heart encoded a 413-amino acid protein with approximately 81% identity to the alpha-variant of mammalian CRF receptor, type 2 (CRF-R2). No evidence could be obtained that the beta-variant of CRF-R2 existed in Xenopus laevis. Binding studies using human embryonic kidney 293 (HEK 293) cells stably transfected with xenopus CRF-R2 showed that the CRF analogues urotensin I, urocortin, and sauvagine were bound with higher affinities than human/rat CRF, xenopus CRF, and ovine CRF. In contrast to human CRF-R1, xenopus CRF-R1 (xCRF-R1) was very selective for different CRF ligands. Urotensin I, urocortin, human/rat CRF, and xenopus CRF were bound with significantly (10-22-fold) higher affinities than ovine CRF (K(D) = 31.7 nM) and sauvagine (K(D) = 51.4 nM). In agreement with these binding data, EC50 values of 39.7 and 1.1 nM were found for sauvagine and for human/rat CRF or xenopus CRF, respectively, when the cyclic AMP production in HEK 293 cells stably transfected with xCRF-R1 was determined.

Amino Acid Sequence↗

PubMed1996

Synthesis and characterization of a photoactivatable analog of corticotropin-releasing factor for specific receptor labeling.

A novel photoactivatable analog of ovine corticotropin-releasing factor (ovine photoCRF) has been synthesized and characterized. A diazirine group, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl residue, was covalently bound to the amino terminus of ovine CRF (oCRF), which was N-terminally extended by a tyrosyl residue for radioactive labeling with 125I. Under mild conditions, photolysis yielded highly reactive carbenes, responsible for the formation of covalent bonds to the CRF receptor. Ovine photoCRF was shown to bind to the high-affinity site of the CRF receptor with a similar Kd value as oCRF. When radioactively iodinated ovine photoCRF (ovine 125I-photoCRF) was covalently linked to rat CRF receptor, type 1 (rCRFR1), permanently transfected into human embryonic kidney (HEK) 293 cells, a highly glycosylated 75-kDa protein was identified with SDS/PAGE. The specificity of ovine 125I-photoCRF was demonstrated by the finding that this analog could be displaced from the receptor by oCRF, but not other unrelated peptides such as vasoactive intestinal peptide. The observed size of the 75-kDa cross-link was in agreement with the molecular weight reported earlier for native CRFR1 from rat brain. Deglycosylation of the 75-kDa cross-link with peptide:N-glycosidase (PNGase) yielded a 46-kDa protein, in agreement with the molecular weight estimated from cDNA coding for rat CRFR1. The developed CRF analog, photoCRF, is expected to facilitate future biochemical and physiological analysis of CRF receptors and--by analogous strategies--of other peptide receptors.

Affinity Labels↗

PubMed1996

Overexpression of a functional NMDA receptor subunit (NMDAR1) in baculovirus-infected Trichoplusia ni insect cells.

For overexpression of the N-methyl-D-aspartate (NMDA) receptor subunit 1b (NMDAR1b), its corresponding cDNA was extended by codons for six histidine residues at the 3'-end, cloned into a baculovirus transfer vector and integrated into the viral genome. Infection of Trichoplusia ni insect cells (High FiveTM cells) with recombinant baculovirus resulted in the production of 126- and 105-kDa NR 1b proteins in the cell membrane fraction. Enzymatic deglycosylation with PNGase F as well as infection of the insect cells in the presence of tunicamycin revealed that the two proteins represented the N-glycosylated and non-glycosylated forms of NMDAR1b, respectively. The recombinant NR1b protein was also identified with immunocytochemical methods employing a monoclonal antibody which recognized the six histidine residues. The affinity of this histidine tag to nickel ions was used for the purification of the NR1b protein. The glycine binding site of the subunit was successfully identified and analyzed with the specific antagonist 5,7-[3-3H]dichlorokynurenate (DCKA). The observed binding characteristics were similar to those obtained for native NMDA receptors. Whereas in electrophysiological measurements a functional NMDA receptor channel could not be found in infected insect cells, its expression was demonstrated in the Xenopus oocyte system after injection of the NMDAR1b cDNA construct.

Animals↗

PubMed1996

Histamine slows the onset of desensitization of rat cortical NMDA receptors.

The effects of histamine on N-methyl-d-aspartate (NMDA) receptor-mediated ion current in cultured rat cortical neurones have been investigated by superfusion of voltage-clamped (-80 mV) cells with 100 mM NMDA and with various concentrations of histamine. In a subpopulation of the neurones tested (30/53), histamine (10-100 mM) enhanced the peak amplitude, and slowed down the onset of desensitization of the NMDA-induced ion currents, both in a concentration-dependent manner. No effects of histamine on the kinetics of recovery from desensitization were detected. In other neurones (21/53), histamine had no effect on the NMDA-induced currents and in two of the neurones tested histamine potentiated NMDA-induced currents without a detectable effect on desensitization kinetics. The results indicate that in a subpopulation of rat cortical neurones histamine potentiates NMDA-induced ion currents by slowing the kinetics of onset of desensitization.

Animals↗

PubMed1996

Serotonin 5-HT2 receptor activation potentiates N-methyl-D-aspartate receptor-mediated ion currents by a protein kinase C-dependent mechanism.

Modulation of N-methyl-D-aspartate (NMDA) receptor-mediated ion currents by serotonin was investigated with a two-electrode voltage clamp technique in Xenopus oocytes injected with rat brain RNA. After a 1-min application of 200 nM serotonin a transient potentiation of the NMDA receptor-mediated ion currents was observed. The serotonin-induced enhancement was mimicked by the protein kinase C activators 1-oleoyl-2-acetyl-sn-glycerol (100 microM) and phorbol 12-myristate 13-acetate (10 nM), whereas the inactive phorbol ester 4-alpha-phorbol 12-myristate 13-acetate (10 nM) had no effect. From these observations it was concluded that protein kinase C was involved in the enhancement of NMDA-induced currents. In agreement with this conclusion, it was found that the serotonin effect was inhibited by the protein kinase C inhibitors sphingosine (1 microM) or staurosporine (1 microM) added 20 min before NMDA application and by oocyte injection of protein kinase C (PKC)-inhibitor peptide (500 ng/oocyte) 1 hr prior to recordings. The serotonin receptor involved was identified as a 5-HT2 receptor subtype by the finding that 200 nM of the selective 5-HT2 receptor agonist alpha-methyl-5-hydroxytryptamine mimicked the potentiation of NMDA-induced ion currents by serotonin. Furthermore, the observed potentiation was significantly reduced by co-application of serotonin with 100 microM of the selective 5-HT2 receptor antagonist ketanserin. These results indicate that 5-HT2 receptors enhance NMDA receptor function via phosphoinositol hydrolysis and subsequent stimulation of PKC.

Animals↗

PubMed1995

Cloning and structure of the gene encoding the human N-methyl-D-aspartate receptor (NMDAR1).

The complete gene encoding the human N-methyl-D-aspartate receptor subunit NR1 (NMDAR1) has been isolated on a single cosmid clone. The gene is composed of 21 exons distributed over a total length of about 31 kb. More than 24 kb were sequenced. Exons 4, 20 and 21 are identical in their amino-acid sequence to those exons that are subject to alternative splicing in rat, indicating that all eight NMDAR1 isoforms found in rat will also be expressed in the human brain. Computer analysis of the pre-mRNA sequence revealed no secondary structures stable enough to explain alternative splicing. We suggest that cell-specific factors control expression of different isoforms. The promoter region contains two perfect copies of the recognition sequence for the Drosophila even-skipped protein, indicating that the developmentally regulated expression of NMDAR1 is controlled by a homeobox protein. The complete cosmid clone covering NMDAR1 was mapped to chromosome 9q34.3-qter by fluorescent in situ hybridization (FISH). The telomeric location is supported by an imperfect (CA)n repeat homologous to a subtelomeric repeat on chromosome 16p.

Alternative Splicing↗

PubMed1995

Glutamate receptor antibodies activate a subset of receptors and reveal an agonist binding site.

Two rabbits immunized with a portion of glutamate receptor (GluR) subunit GluR3 (amino acids 245-457) exhibited seizure-like behaviors, suggesting that antibodies to GluR3 may modulate neuronal excitability. Using whole-cell recording, rabbit GluR3 antisera were tested on cultured fetal mouse cortical neurons. In a subset of kainate-responsive neurons, miniperfusion of antisera and IgG evoked currents that were blocked by CNQX. Immunoreactivity to synthetic peptides prepared to subregions GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395) was present in both rabbit sera. Peptide GluR3B, but not GluR3A, specifically blocked antisera- and IgG-evoked currents. Similar receptor activation and anti-GluR3 reactivity was present in sera from patients with active Rasmussen's encephalitis, an intractable pediatric epilepsy. Thus, antibodies to GluR3 define a region involved in agonist binding and specific receptor activation. These data suggest that antibodies to neuronal receptors can function as agonists and that autoantibodies to GluRs may be highly specific neurotoxicants in some neurological diseases.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1993

Characterization of the NR1, NR2A, and NR2C receptor proteins.

N-Methyl-D-aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies. The antibodies were directed against NR1-A, NR2A-N1, and NR2C-N1, representing N-terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1-E, NR2A-C1, and NR2C-C1, derived from C-terminal sequences of these subunits. The anti-NR1-D antibody was raised against the putative internal loop of NR1. A size of 118 kDa was found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti-NR1-D and -NR1-E, but not anti-NR1-A. With the anti-NR1-A antibody, a 125-kDa protein was discovered that may represent a glutamate receptor not yet characterized. NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively. Enzymatic N-deglycosylation generated a 97-kDa protein from NR1, a 105-kDa protein from the 125-kDa protein, a 162-kDa protein from NR2A, and a 127-kDa protein from NR2C. In contrast to the deglycosylation product of the NR2A, the 97- and 127-kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data. These products may represent truncated proteins. The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum. In contrast, NR2C was solely found in the cerebellum. The 125-kDa protein was highest in the cerebellum and cortex.

Amino Acid Sequence↗

PubMed1993

Immortalization of a fetal rat brain cell line that expresses corticotropin-releasing factor mRNA.

By introducing the SV40 T antigen under the control of promoter sequences from the gene encoding the rat corticotropin-releasing factor (CRF) into primary cultures from fetal rat brain, a stable cell line was established that expresses the SV40 T antigen and transcribes its endogenous CRF gene. The cell line exhibits neuronal properties, as demonstrated by positive immunostaining with antibodies against neuronal markers, and it provides a useful system for the detailed analysis of the transcriptional regulation of the neuropeptide gene at an early stage of development. Promoter activities of the CRF 5'-flanking region were tested in transient expression assays after transfection of the cell line with different fusion constructs with CRF promoter regions linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Basal activity of the promoter was determined by DNA sequences between positions -269 and -222 upstream of the transcriptional start site and showed weak induction upon treatment with forskolin.

Animals↗