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J Rossier

Publications and source records attributed to J Rossier.

At least 109 records · Page 6Linked to original sources
PubMed1991

Reduction of desensitization of a glutamate ionotropic receptor by antagonists.

The glutamate receptor channel subtype that responds to both quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) was expressed in Xenopus oocytes injected with rat cerebral cortex mRNA. Voltage-clamp current responses to QA, AMPA, and glutamate (GLU) exhibited a rapid increase followed by a decrease to a desensitized steady state (DS). Perfusion with high agonist concentrations produced smaller DS responses than perfusion with low concentrations. During the DS, the current was increased by lowering of the concentration of agonist or by application of low concentrations of a competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX). This paradoxical increase of the agonist-induced currents during the DS was also observed in cultured Purkinje cells with another competitive antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). Dose-response curves obtained in oocytes were bell shaped, with a negative slope for high concentrations of QA. DNQX shifted these bell-shaped curves to the right. Together, these results indicate that the agonists are able to reversibly inhibit the AMPA receptor. The classical desensitization model of Katz and Thesleff [J. Physiol. (Lond.) 138:63-80 (1957)] cannot account for our observations.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1990

A 15 kDa proteolipid found in mediatophore preparations from Torpedo electric organ presents high sequence homology with the bovine chromaffin granule protonophore.

Upon SDS PAGE of isolated mediatophore, an acetylcholine-translocating protein, a doublet at 15 kDa was identified. Amino acid sequencing after CNBr cleavage gave a 17 residue-long peptide completely homologous with a sequence of the proton-translocating proteolipid from bovine chromaffin granules. A 51-mer oligodeoxynucleotide corresponding to this sequence was used to screen a library of electric lobe cDNAs constructed in lambda Zap II. A positive recombinant clone was isolated and found to encode the complete sequence of a 15.5 kDa protein highly homologous to the bovine chromaffin or yeast vacuolar ATPase proteolipid. In vitro translation of sense RNA transcripts of the clone indeed yielded a single 15 kDa proteolipid. Northern blot analysis showed that the 1.3 kb mRNA encoding this protein is significantly expressed in nervous tissues but not in electric organ or liver of Torpedo marmorata.

Acetylcholine↗
PubMed1990

Posttranslational glutamylation of alpha-tubulin.

The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of alpha-tubulin can be posttranslationally labeled after incubation of cells with [3H]acetate or [3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the gamma-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of alpha-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.

Amino Acids↗
PubMed1990

Detection of synenkephalin, the amino-terminal portion of proenkephalin, by antisera directed against its carboxyl terminus.

Synenkephalin (SYN), the nonopioid amino-terminal portion of proenkephalin (PRO), is stable and well conserved in mammals and therefore a promising marker for PRO systems. We immunized rabbits with synthetic [Tyr63]SYN(63-70)-octapeptide, coupled by glutaraldehyde to bovine serum albumin. In radioimmunoassay (RIA) using antiserum no. 681, [Tyr63]SYN(63-70)-octapeptide as standard, and 125I-[Tyr63]SYN(63-70)-octapeptide as tracer, the IC50 was approximately 51 fmol/100-microliters sample at equilibrium or 12 fmol/100 microliters in disequilibrium, and the sensitivity was approximately 3 fmol/100 microliters. Cross-reactivity of the assay was 100% with [Cys63]SYN(63-70)-octapeptide and with bovine adrenal 8.6-kilodalton peptide digested with trypsin and carboxypeptidase B, but less than 0.1% with transforming growth factor-alpha, less than or equal to 2 x 10(-6) with Leu-Leu-Ala [SYN(68-70)-tripeptide], and much less than 10(-6) with all other peptides tested. Therefore in RIA this antiserum is specific for the free carboxyl terminus of SYN. Because the peptide detected after enzyme digestion is the complete SYN(63-70)-octapeptide, we refer to the RIA as an assay for SYN(63-70). Tissue extracts were made in 1 M acetic acid, dried, reconstituted in Tris-CaCl2, and digested sequentially with trypsin plus carboxypeptidase B. Extracts from bovine corpus striatum gave SYN(63-70) RIA dilution curves parallel to the standard curve both before and after digestion. Digestion increased the amount of immunoreactive SYN(63-70) in striatum by a factor of 1.5-2.0. The ratio of total immunoreactive [Met5]enkephalin to total immunoreactive SYN(63-70) (after sequential digestion) was approximately 6:1. At least 90% of the immunoreactive SYN(63-70) in extracts of bovine caudate nucleus eluted from Sephadex G-100 with an apparent molecular weight equal to that of bovine PRO(1-77). Using the new RIA we were able to detect and characterize SYN processing for the first time in extracts of whole rat brain, human globus pallidus, and human pheochromocytoma. Results in these tissues were similar to those in cattle, in that most stored SYN had been processed to a free carboxyl terminus. Since the C-terminal octapeptide of SYN is practically identical in all known mammalian PRO, antiserum no. 681 should be useful for detecting, measuring, and purifying SYN from various mammals, including human beings.

Adrenal Gland Neoplasms↗
PubMed1989

Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans.

The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Isolation of dermenkephalin from amphibian skin, a high-affinity delta-selective opioid heptapeptide containing a D-amino acid residue.

The predicted amino acid sequence of the biosynthetic precursor of dermorphin, a highly potent and nearly specific mu-opioid peptide from amphibian skin, contains four repeats of the dermorphin progenitor sequence and one single copy of a different heptapeptide sequence. We have developed a specific enzyme immunoassay and used synthetic peptides to detect and purify the new predicted heptapeptide (2.4 micrograms/g dry skin) from the skin of the Phyllomedusa sauvagei frog from which dermorphin was originally isolated. The identity of the novel pro-dermorphin related peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, was established by co-chromatography with synthetic peptides on reverse-phase HPLC, immunological analysis, gas-phase sequencing, mass spectrometry and by pharmacological assays. Opioid-binding assays in vitro demonstrated that both the natural and synthetic heptapeptides displayed exceptionally high selectivity and affinity towards the delta-opioid receptors. Because of its origin and its delta-opioid (enkephalin) activity and specificity, this novel D-amino acid containing peptide is named dermenkephalin.

Acetylcholinesterase↗
PubMed1989

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes.

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.

Animals↗
PubMed1989

Synthesis and benzodiazepine receptor affinities of rigid analogues of 3-carboxy-beta-carbolines: demonstration that the benzodiazepine receptor recognizes preferentially the s-cis conformation of the 3-carboxy group.

1H-Indolo[3',2':4,5]pyrido[3,2-b]-2-penten-5-olide (6) and 1H,5H-indolo[3',2'-c]-6,7-dihydro-2-pyridone (7), rigid analogues of methyl 4-ethyl-beta-carboline-3-carboxylate (8) and N-methyl-4-ethyl-beta-carboline-3-carboxamide (9), respectively, were synthesized and their in vitro binding affinities to the central type benzodiazepine receptors were compared. The IC50 values of 6 and 8 were approximately equivalent (42 and 27 nM, respectively). The amide derivative 9, for which theoretical energy calculations indicate that the s-trans carbonyl conformation is the preferred one, displayed very low affinity (IC50 greater than 10(4) nM). However, when the carbonyl group of 9 was forced to adopt the s-cis conformation as in lactam 7, binding to the benzodiazepine receptor was largely restored (IC50 = 150 nM), indicating that the s-cis carboxy conformation at C-3 of beta-carbolines is preferentially recognized by this receptor. In vivo, compound 6 showed neither convulsant, proconvulsant, nor anticonvulsant activity in mice. Moreover, 6 did not antagonize methyl beta-carboline-3-carboxylate induced convulsions in mice. This lack of activity of 6 was attributed to its inability to cross the blood-brain barrier since no significant displacement of [3H]Ro 15-1788 from mouse brain benzodiazepine receptors by 6 could be observed in vivo.

Animals↗
PubMed1988

Demonstration of the partial agonist profiles of Ro 16-6028 and Ro 17-1812 in mice in vivo.

Benzodiazepine receptor occupancy by the full agonist, diazepam, and by the two putative partial agonists, Ro 16-6028 and Ro 17-1812, was measured by inhibition of in vivo [3H]Ro 15-1788 binding in mouse brain and was correlated with their pharmacological effects. The anticonvulsant effects of Ro 16-6028, Ro 17-1812 and diazepam (ED50 values) appeared at receptor occupancies of 40, 20 and less than 5%, respectively. Moreover, at the highest measurable receptor occupancy (90-100%), Ro 16-6028 and Ro 17-1812 did not induce any rotarod deficit whereas a complete deficit was observed with diazepam at 35% receptor occupancy.

Animals↗
PubMed1988

Selective cleavage of proenkephalin-derived peptides (less than 23,300 daltons) by plasma kallikrein.

The ability of human plasma kallikrein to hydrolyze several proenkephalin-derived peptides has been studied, including the synthetic peptides BAM 12P and peptides E, F, and B as well as synenkephalin-containing peptides (8.6, 18.2, and 23.3 kDa) purified from bovine adrenal medulla chromaffin granules. All the identified cleavages occurred either COOH-terminal to or between pairs of basic amino acids, with plasma kallikrein recognizing Lys-Lys, Lys-Arg, and Arg-Arg as processing signals. Moreover, plasma kallikrein was found to cleave at the COOH terminus of the basic pairs of amino acids preceding enkephalin sequences thereby releasing the biologically active form of the peptide with the free NH2-terminal Tyr needed for receptor recognition.

Adrenal Medulla↗
PubMed1988

Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.

Chemical Precipitation↗
PubMed1988

On the use of polyethylenimine as a carrier for protein sequencing: comparison with polybrene.

In the gas-liquid phase automated protein sequencer, polyethylenimine was used as a hydrophilic entrapping polymer. Glass fiber filters soaked in 0.3% solution of polyethylenimine were used. Sperm whale myoglobin, beta-lactoglobulin, and several peptides with basic or acidic pI were sequenced. Loads from 20 to 26,000 pmol were tested. Initial and repetitive yields compare favorably to those obtained with polybrene-coated glass fiber filters. Recovery of individual amino acids shows that none gave a particularly low yield, in contrast with the low recovery of Arg, Trp, His, Glu, and Asp when polybrene was used. The usual artefacts were greatly diminished and even disappeared as in the case of N,N'-diphenylurea. Substitution of polyethylenimine for polybrene sped up the analysis because the precycling employed to condition polybrene-coated glass fiber filters was no longer necessary. In conclusion polyethylenimine appears superior to polybrene for sequencing protein and peptides.

Amino Acid Sequence↗
PubMed1988

In vivo binding of (3H)Ro 15-1788 in mice: comparison with the in vivo binding of (3H)flunitrazepam.

Benzodiazepine binding sites have generally been labelled with benzodiazepine agonists: (3H)flunitrazepam and (3H)diazepam in vivo. We studied the in vivo binding of the antagonist (3H)Ro 15-1788 in mice and compared it to the in vivo binding of (3H)flunitrazepam. For this in vivo labelling, mice were injected with labelled and unlabelled ligands. Animals were then sacrificed and bound radioactivity was measured after homogenization of the excised brain and filtration of the homogenate. (3H)Ro 15-1788 is a better tool than (3H)flunitrazepam for in vivo labelling of benzodiazepine receptors since 1) it labels specifically the central type binding sites, 2) injection of 4 times less (3H)Ro 15-1788 (50 microCi/kg) than (3H)flunitrazepam (200 microCi/kg) produced the same amount of bound radioactivity, 3) 70-90% of the total (3H)Ro 15-1788 present in the brain is membrane bound instead of 45-55% with (3H)flunitrazepam, 4) maximal binding of (3H)Ro 15-1788 is reached within 3 min, 5) only 5% of the membrane bound (3H)Ro 15-1788 is nonspecific instead of 15% for (3H)flunitrazepam.

Animals↗
PubMed1988

Liberation of enkephalins from enkephalin-containing peptides by brain endo-oligopeptidase A.

Endo-oligopeptidase A, highly purified from the cytosol fraction of bovine brain by immunoaffinity chromatography, has been characterised as a thiol endopeptidase. This enzyme, known to hydrolyse the Phe5-Ser6 bond of bradykinin and the Arg8-Arg9 bond of neurotensin has been shown to produce, by a single cleavage, [Leu]enkephalin or [Met]enkephalin from small enkephalin-containing peptides. Enkephalin formation could be inhibited in a concentration dependent manner by the alternative substrate bradykinin. The optimal substrate size was found to be 8-13 amino acids, with enkephalin the only product released from precursors in which this sequence is immediately followed by a pair of basic residues. However, the specificity constants (kcat/Km) obtained for endo-oligopeptidase A hydrolysis of bradykinin, neurotensin and dynorphin B are of the same order. Taken together, these results indicate that the substrate amino acid sequence is not the only factor determining the cleavage site of this enzyme. Finally, endo-oligopeptidase A and metalloendopeptidase EC 3.4.24.15 are two different enzymes. The latter is not able to liberate enkephalins from metorphamide and dynorphin.

Amino Acid Sequence↗
PubMed1988

In vivo binding of beta-carbolines in mice: regional differences and correlation of occupancy to pharmacological effects.

beta-Carbolines are competitive ligands of central type benzodiazepine receptors and have been reported to display either agonist, inverse agonist, or antagonist activities in vivo. We studied the in vivo inhibition of [3H]Ro 15-1788 binding in mice by various beta-carbolines of different pharmacological profiles and found that they were all more potent in the cerebellum than in the cortex and hippocampus; their ID50 values (dose inhibiting 50% of the specific binding of [3H]Ro 15-1788) were 3 to 7 times lower in the cerebellum than in the hippocampus. The ID50 of the triazolopyridazine CL 218,872 was 2.3 times lower in the cerebellum than in the hippocampus. Thus, regional differences do not seem to explain pharmacological profile. Correlation of receptor occupancy with pharmacological effects showed that high receptor occupancy (40%) was needed to obtain the convulsant effects of the inverse agonist methyl-beta-carboline-3-carboxylate whereas intermediate receptor occupancy (30%) led to the proconflict effects and very low receptor occupancy (less than 5%) to the facilitating effects in learning and memory tasks. We also found that the selective antagonist of the sedative effects of benzodiazepines 3-(methoxycarbonyl)-amino-beta-carboline, even at high doses (20 mg/kg), did not occupy more than 70% of the benzodiazepine receptors.

Animals↗
PubMed1988

Photoaffinity labeling of opioid delta receptors with an iodinated azido-ligand: [125I][D-Thr2,pN3Phe4,Leu5]enkephalyl-Thr6.

The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) was iodinated and purified by high performance liquid chromatography. Monoiodo-azido-DTLET displayed a high affinity (KD = 15 nM) and is selective (Kl mu/Kl delta = 9.8) for rat brain delta opioid receptors (for comparison, the corresponding values for tritiated azido-DTLET are KD = 1.66 nM and Kl mu/Kl delta = 27). On rat brain sections, the anatomical distribution of [125I]azido-DTLET binding sites revealed by autoradiography corresponds to that of delta receptors. On rat brain membrane homogenates and NG108-15 hybrid cells, UV irradiation of the receptor-ligand complex results in the irreversible binding to membrane proteins of 14% of the bound radioactivity Gel electrophoresis of [125I]azido-DTLET-labeled proteins followed by autoradiography shows a different pattern in rat brain and NG108-15 cells. In rat brain, labeling of two of these proteins, with molecular weights of 44,000 and 34,000, was inhibited by 30 nmol/liter of nonradioactive DTLET, a delta-selective ligand but not by the same concentration of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-selective ligand. In NG108-15 cells, this 44-kDa protein was not visualized; the main band was at 33 kDa and disappeared in the presence of levorphanol.

Affinity Labels↗