Biomedical subjects

P Nicolas

Publications and source records attributed to P Nicolas.

At least 109 records · Page 6Linked to original sources

PubMed1993

Molecular cloning of a cDNA encoding the precursor of adenoregulin from frog skin. Relationships with the vertebrate defensive peptides, dermaseptins.

Adenoregulin has recently been isolated from Phyllomedusa skin as a 33 amino acid residues peptide which enhanced binding of agonists to the A1 adenosine receptor. In order to study the structure of the precursor of adenoregulin we constructed a cDNA library from mRNAs extracted from the skin of Phyllomedusa bicolor. We detected the complete nucleotide sequence of a cDNA encoding the adenoregulin biosynthetic precursor. The deduced sequence of the precursor is 81 amino acids long, exhibits a putative signal sequence at the NH2 terminus and contains a single copy of the biologically active peptide at the COOH terminus. Structural and conformational homologies that are observed between adenoregulin and the dermaseptins, antimicrobial peptides exhibiting strong membranolytic activities against various pathogenic agents, suggest that adenoregulin is an additional member of the growing family of cytotropic antimicrobial peptides that allow vertebrate animals to defend themselves against microorganisms. As such, the adenosine receptor regulating activity of adenoregulin could be due to its ability to interact with and disrupt membranes lipid bilayers.

Amino Acid Sequence↗

PubMed1993

Quantitative two-dimensional NMR study of dermenkephalin, a highly potent and selective delta-opioid peptide.

Dermenkephalin, H-Tyr-(D)Met-Phe-His-Leu-Met-Asp-NH2, a highly potent and selective delta-opioid peptide isolated from frog skin, was studied in DMSO-d6 solution by two-dimensional nmr spectroscopy, including the determination of NH temperature coefficients, the evaluation of 3J coupling constants from phase-sensitive correlated spectroscopy (COSY) and the volumes of nuclear Overhauser effect (NOE) correlations. The two-dimensional NOE spectroscopy (NOESY) spectrum of dermenkephalin revealed sequential, medium-, and long-range effects. To put this information on a quantitative basis, special attention was devoted to J cross-peak suppression, quantification of the NOE volumes and analysis of the overlaps, normalization of the NOEs against diagonal peaks and H beta beta' geminal interactions. Although most of the dihedral angles deduced from the 3JN alpha coupling constants together with several Ni alpha i and alpha iNi+1 NOEs pointed to a partially extended peptide backbone, several NiNi+1 NOEs and beta iNi+1 interactions argued in favor of a folded structure. Moreover, several long-range correlations of strong intensities were found that supported a close spatial proximity between the side chains of D-Met2 and Met6, Tyr1 and His4, Tyr1 and Asp7, and His4 and the C-terminal amide group. In Phe, the g- rotamer in the side chain is deduced from the 3J alpha beta coupling constants and alpha beta and N beta NOE correlations. Whereas the amide proton dependency was not indicative of stable hydrogen bonds, the nonuniform values of the temperature coefficient may reflect an equilibrium mixture of folded and extended conformers. The overall data should provide realistic starting models for energy minimization and modelization studies.

Amino Acid Sequence↗

PubMed1992

Characterization of receptors for thyrotropin-releasing hormone-potentiating peptide on rat anterior pituitary membranes.

Previous studies (Bulant, M., Delfour, A., Vaudry, H., and Nicolas, P. (1988) J. Biol. Chem. 263, 17189-17196; Bulant, M., Roussel, J. P., Astier, H., Nicolas, P., and Vaudry, H. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4439-4443) have shown that post-translational processing of rat thyrotropin-releasing hormone prohormone (pro-TRH) generates, besides thyrotropin-releasing hormone (TRH), a connecting decapeptide corresponding to prepro-TRH-(160-169), i.e. Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr. This peptide, which is named TRH-potentiating peptide (Ps4), is co-localized with TRH in the median eminence nerve endings and is involved in potentiation of the action of TRH on thyrotropin hormone release by pituitary in vitro and in vivo. To characterize the receptor(s) for TRH-potentiating peptide in the pituitary, a highly potent and metabolically stable derivative of Ps4, [I-Tyr0]Ps4, was radioiodinated. Binding of [125I-Tyr-0]Ps4 to rat pituitary membrane homogenates was specific, saturable, reversible, and linear with membrane protein concentration. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high affinity binding sites (Kd = 0.22 nM; Bmax = 517 fmol/mg of membrane proteins). Several naturally occurring neuropeptides and hormones, including TRH, did not compete with [125I-Tyr0]Ps4 in the binding, which suggests the binding sites are specific to Ps4. Using C-terminal deletion analogs of [Tyr0]Ps4, we further showed the critical role the C-terminal residues Thr10, Val9, and Asp8 play in conferring high binding affinity and selectivity. Binding site tissue distribution and cross-reactivity binding studies suggest that the action of TRH-potentiating peptide is mediated through interaction with a specific pituitary cell-surface receptor which differ from those for TRH. [I-Tyr0]Ps4 reported in this paper, through its high binding affinity and specificity, its very low nonspecific binding, its high resistance to enzymatic degradation, and its high potentiating action in vitro should allow further progress in understanding the in vivo physiological function of Ps4.

Amino Acid Sequence↗

PubMed1992

Isolation and amino acid sequence of the TRH-potentiating peptide from bovine hypothalamus.

A neuropeptide termed TRH-potentiating peptide, which potentiates TRH-evoked thyrotropin secretion by antehypophysis in vitro, was isolated from an acetonic powder of bovine hypothalamus. The peptide was purified to homogeneity by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography and reverse phase high performance liquid chromatography. The complete amino acid sequence of the decapeptide was determined as Ser-Phe-Pro-Trp-Met-Glu-Ser-Asp-Val-Thr by automated Edman degradation with a solid-phase sequencer. Bovine TRH-potentiating peptide is structurally identical to Ps4, a decapeptide which was deduced from the cDNA encoding the rat TRH precursor. This study provides for the first time a direct chemical evidence for the existence of non-TRH peptides originating from posttranslational processing of the TRH precursor in vivo.

Amino Acid Sequence↗

PubMed1992

Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes.

Dermaseptin, a 34 amino-acid residue antimicrobial polypeptide [Mor, A., Nguyen, V. H., Delfour, A., Migliore-Samour, D., & Nicolas, P. (1991) Biochemistry 30, 8824-8830] was synthesized and selectively labeled at its N-terminal amino acid with either 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), rhodamine, or fluorescein. The fluorescent emission spectra of the NBD-labeled dermaseptin displayed a blue-shift upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. Titrations of solutions containing NBD-labeled dermaseptin with SUV composed of zwitterionic or acidic phospholipids were used to generate binding isotherms, from which were derived surface partition constants of (0.66 +/- 0.06) x 10(4) M-1 and (2.8 +/- 0.3) x 10(4) M-1, respectively. The shape of the binding isotherms, as well as fluorescence energy transfer measurements, suggests that some aggregation of membrane-bound peptide monomers occurs in acidic but not in zwitterionic vesicles. The preferential susceptibility of the peptide to proteolysis when bound to zwitterionic but not to acidic SUV suggests that these aggregates might then penetrate a relatively short distance into the hydrophobic region of the acidic membrane. Furthermore, the results provide good correlation between the peptide's strong binding and its ability to permeate membranes composed of acidic phospholipids, as revealed by a dissipation of diffusion potential and a release of entrapped calcein from SUV.

Amino Acid Sequence↗

PubMed1992

The aspartic acid in deltorphin I and dermenkephalin promotes targeting to delta-opioid receptor independently of receptor binding.

Recent studies on the highly potent and selective delta-opioid agonists demenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) suggested that key structural features necessary for specific targetting to the delta-opioid receptor are located within the C-terminal halves of these naturally occurring heptapeptides. To investigate the contribution of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin to the delta-addressing ability of the C-terminal ends, fourteen analogs were synthesized and assessed for their ability to bind to mu and delta-opioid receptors in rat brain membrane homogenates. Results showed that i/ although the tetrapeptide C-terminus of dermenkephalin and deltorphin I differ in amino acid composition, they play a similar role in specifying correct addressing of these peptides to the delta-receptor, ii/ the negatively charged side chain of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin is not involved in binding contact at the delta-receptor site, nor in maintaining a delta-bioactive folding of the peptides, iii/ these side chains are, in contrast, functionally or structurally required to confer high delta-selectivity by preventing mu-site recognition and/or binding.

Amino Acid Sequence↗

PubMed1992

Sensitive determination of josamycin and rokitamycin in plasma by high-performance liquid chromatography with fluorescence detection.

Rokitamycin and josamycin were successfully derivatized with dansylhydrazine in 20 min at 60 degrees C. Rokitamycin and josamycin levels were determined in plasma after ion-pair extraction into hexane-isoamyl alcohol with lauryl sulphate and precolumn derivatization. Resolution was obtained by liquid chromatography with fluorescence detection (352/537 nm) in 12 min. The limit of detection was 20 ng/ml macrolide starting from 1 ml of plasma, and linearity was demonstrated between 50 and 400 ng/ml. Inter-run coefficients of variation were 10.2% at 100 ng/ml and 9.1% at 300 ng/ml. The system was reliably used for pharmacokinetic studies in plasma.

Chromatography, High Pressure Liquid↗

PubMed1992

Pharmacokinetics of ibuprofen enantiomers after single and repeated doses in man.

The pharmacokinetic parameters of ibuprofen enantiomers after a single 600 mg dose and repeated 3 x 400 mg doses of Nurofen were determined in 12 healthy volunteers. Terminal half-lives were similar for both enantiomers, but plasma levels of S-ibuprofen were higher than those of R-ibuprofen, due to the chiral inversion and differences in distribution and metabolism. Comparison of maximal concentrations and areas under the concentration vs time curves between the first and last doses for each enantiomer indicated linear pharmacokinetics with no time-dependency. A large inter-individual variability in the ratio of S- to R-ibuprofen average concentrations at steady-state was observed (mean +/- SD 1.89 +/- 0.89) and probably accounts for the known lack of correlation between racemic ibuprofen concentrations and therapeutic efficacy.

Adult↗

PubMed1992

Enter a new post-translational modification: D-amino acids in gene-encoded peptides.

The post-translational processing of peptides plays a key role in conferring biological activity on those peptides. Recently, ribosomally made peptides that contain D-amino acids at specific positions have been discovered in microorganisms as well as in vertebrates and invertebrates. This points to yet another strategy of circumventing stereochemical limitations imposed by the genetic code and conveying biological activity to otherwise inert molecules.

Amino Acid Sequence↗

PubMed1992

Amiodarone decreases cardiac beta-adrenoceptors through an antagonistic effect on 3,5,3' triiodothyronine.

Because surprising similarities exist between the cardiac effects of amiodarone and those observed during hypothyroidism, we tested three different mechanisms of action in rats to determine whether amiodarone might act by inducing a tissular hypothyroidism: (a) a decrease in thyroid secretion, (b) an inhibiting effect on the conversion of thyroxine (T4) to 3,5,3' triiodothyronine (T3), and (c) a direct antagonistic effect on the cellular action of T3. Five groups of rats, treated orally for 7 or 13 days, were studied: I, control (0.5 ml saline for 7 or 13 days, n = 14); II, amiodarone (50 mg/kg for 7 days, n = 5); III, iopanoic acid (100 mg/kg for 13 days, n = 7); IV, control + T3 (0.5 ml saline for 13 days and 0.5 mg/kg T3 for the last 6 days, n = 5); V, amiodarone + T3 (amiodarone 50 mg/kg for 13 days and 0.5 mg/kg T3 for the last 6 days, n = 5). Cardiac beta-adrenoceptor density (CBARD) and heart rate (HR) were the two endpoint parameters investigated. Thyroid status was evaluated by serum thyrotropin (TSH), T4, T3, rT3 concentrations and liver type I 5'-deiodinase (5'D-I) activity. Amiodarone (group II) decreased CBARD (-22%, p less than 0.05) without altering thyroid secretion and T3 serum level, whereas 5'D-I was strongly inhibited (-90%, p less than 0.01). Iopanoic acid had no effect on CBARD and HR, but deeply inhibited 5'D-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiodarone↗

PubMed1992

In vivo effects of amiodarone on cardiac beta-adrenoceptor density and heart rate require thyroid hormones.

To assess if the anti-beta-adrenergic effect and the bradycardia induced by amiodarone were mediated by thyroid hormone, we investigated these effects of amiodarone in euthyroid and hypothyroid rats. We studied control rats, thyroidectomized rats, control rats treated with amiodarone (50 mg/kg for 8 days), and thyroidectomized rats treated with amiodarone. At the end of the treatment, free thyroid hormone levels (FT4 and FT3) were determined, and cardiac beta-receptor density (Bmax) and affinity (Kd) were assayed by using (-)-[125I]iodocyanopindolol as radioligand. Resting heart rate (rHR) was also assessed every day in control and thyroidectomized rats, before and after amiodarone. In hypothyroid rats, in which free thyroxine (FT4) was not detectable and free 3,5,3'-triiodothyronine (FT3) was only 16% that of euthyroid rats, Bmax (14.1 +/- 2.5 fmol/mg, n = 7) and rHR (259 +/- 9.7 beats/min, n = 6) were significantly lowered compared with euthyroid rats (Bmax:18.4 +/- 3.4 fmol/mg, n = 7; rHR:277 +/- 4.1 beats/min, n = 5). Amiodarone treatment decreased Bmax (13.6 +/- 2.9 fmol/mg, n = 8) and rHR (252 +/- 5.5 beats/min, n = 5) only in euthyroid rats and did not produce significant cardiac effects in hypothyroid rats. (Values are given as mean +/- SD.) We conclude that a minimum serum thyroid hormone concentration is a necessary condition for amiodarone to produce some of its cardiac effects. An antagonistic reaction to thyroid hormones at the cellular level can be postulated as a mechanism of the cardiac anti-beta-adrenergic action of amiodarone.

Amiodarone↗

PubMed1992

Pharmacokinetics and absolute rectal bioavailability of hydrocortisone acetate in distal colitis.

The hydrocortisone pharmacokinetic profiles of hydrocortisone acetate foam (Proctocort) administered rectally was assessed in healthy volunteers and patients with ulcerative colitis or X-irradiation colitis. Endogenous production of hydrocortisone was suppressed by dexamethasone. Comparison of these data with those obtained after intravenous administration enabled assessment of absolute bioavailability, which was 30.0 +/- 15.1% in healthy volunteers vs. 16.4 +/- 14.8% in patients (P = 0.09). Maximal concentrations of hydrocortisone were also decreased in patients, 277 +/- 215 nmol/L vs. 610 +/- 334 nmol/L (P = 0.03). There was a nonsignificant tendency to faster absorption of hydrocortisone in patients vs. healthy volunteers, as the times to peak concentration were, respectively, 2.5 +/- 1.2 h vs. 2.8 +/- 0.8 h (P = 0.64), and the mean absorption times were 1.96 +/- 1.45 h vs. 2.54 +/- 1.62 h (P = 0.46). Thus, rectal inflammation resulted in a lower absorption of hydrocortisone. In addition systemic plasma levels remained in the physiological range, so that only minor side effects are to be expected.

Administration, Rectal↗

PubMed1992

Pharmacokinetics of vidarabine in the treatment of polyarteritis nodosa.

The pharmacokinetics of vidarabine were studied in 8 patients with polyarteritis nodosa related to hepatitis B virus infection. The drug was administered by continuous infusion for three weeks at doses of 15 (1 week) and 7.5 (2 weeks) mg/kg per day, during which time 15 plasma exchanges were performed. Plasma was assayed for vidarabine and its principal metabolite, hypoxanthine arabinoside by high pressure liquid chromatography. Vidarabine was not detected in the plasma of any patients. Hypoxanthine arabinoside levels were used to evaluate vidarabine kinetics. The serum levels of hypoxanthine arabinoside ranged from 3.6 to 21.5 mg/l. The mean elimination half-life (+/- SD) was 3.0 +/- 1.7 h. The plasma clearance (mean +/- SD) was 195 +/- 270 ml/min when the dose was 7.5 mg/kg per day and 66.3 +/- 47 ml/min for a 15 mg/kg per day/dose (NS). Except for the elimination half-life, these results were not fully consistent with those observed in other studies. The influence of multiple plasma exchanges on vidarabine kinetics is limited and dosage adjustment is not required based on the continuous infusion of vidarabine.

Humans↗

PubMed1992

Pharmacokinetics of glafenine and glafenic acid in patients with cirrhosis, compared to healthy volunteers.

Pharmacokinetic parameters were evaluated in 12 patients with alcoholic cirrhosis and 12 healthy volunteers after a single 400 mg oral dose of glafenine. Glafenine (G) and its major active metabolite glafenic acid (GA) were measured at regular intervals using a specific high performance liquid chromatographic method. Glafenine absorption was significantly delayed in cirrhotic patients (CP) (Tmax = 2.8 +/- 1.3 hvs 1.5 +/- 0.4 h, p less than 0.01) and was dramatically reduced in 3 patients. The large hepatic 'first pass' effect observed in healthy volunteers was markedly reduced in CP (ratio Cmax GA/Cmax G = 3.6 +/- 2.9 vs 18.9 +/- 9.8, p less than 0.001; ratio areas under the curves AUC GA/AUC G = 2.3 +/- 2.3 vs 18.2 +/- 11.2, p less than 0.001). The elimination half-life of G was prolonged in the CP (13.0 +/- 13.1 h vs 1.5 +/- 0.5 h, p less than 0.01). In CP, GA elimination half-life was increased (12.0 +/- 13.4 h vs 4.3 +/- 1.3 h, NS) but the difference did not reach statistical significance because of large variability. The significant rise of G plasma concentrations (Cmax = 2.2 +/- 2.1 mg/L vs 0.7 +/- 0.2 mg/L, p less than 0.05) and its longer half-life would lead to an accumulation if the usual dosage regimen was prescribed for CP and could result in nephrotoxicity. On the other hand, lower dosage would be ineffective because only GA is active and nephrotoxic. Hence, G should be given with great caution to CP.

Administration, Oral↗

PubMed1992

Pattern of thyrotropin-releasing hormone secretion from the adult and neonatal rat pancreas: comparison with insulin secretion.

In the present study we examined the secretory pattern of pancreatic TRH. TRH is synthesized in the islets of Langerhans as a high mol wt prohormone, which is then processed to give TRH and several putative pro-TRH fragments. The mature TRH was localized in insulin-containing cells of developing rat pancreas, marked by elevated TRH concentrations. The biological role of pancreatic TRH in the regulation of major pancreatic hormones secretion is still poorly understood. We, therefore, studied TRH release from isolated perfused rat pancreas and from neonatal and adult islets in the absence and presence of glucose and arginine to investigate regulation of TRH secretion compared to that of insulin secretion. The secretory pattern of TRH was the opposite of that of insulin in adult pancreas, but was similar to that of insulin in the developing pancreas, suggesting the presence of a subpopulation of beta-cells with peculiar age-dependent topololgy and/or different intracellular transport for TRH. Additionally, antibodies to two pro-TRH-connecting peptides, pro-TRH-(160-169) and pro-TRH-(178-199), were used to characterize the processing in the developing and the adult rat pancreas. There were parallel age-dependent decreases in TRH and both connecting peptides. Interestingly enough, only pro-TRH-(178-199) was selectively released, suggesting that some of the pro-TRH fragments may also assume biological effects. The establishment of these parameters will allow appropriate screening of the biological response to TRH.

Aging↗

PubMed1992

Functional and structural damage in Leishmania mexicana exposed to the cationic peptide dermaseptin.

The effect of dermaseptin (DS), a 34 amino acid residue cationic peptide isolated from Phyllomedusa sauvagii skin, has been studied on promastigotes of Leishmania mexicana growing in vitro. Within 5 min of incubation in the presence of DS, the flagellated parasites lost their motility. DS inhibited promastigote growth by 50% at a concentration of 3 microM and by 100% at 10 microM. Immunocytochemical, freeze fracture, label fracture and electron microscopic observations have shown that the amphipathic peptide generates perturbations of the lipid bilayer leading to altered permeability of the surface membrane and death of the parasite.

Amphibian Proteins↗

PubMed1991

Dermenkephalin and deltorphin I reveal similarities within ligand-binding domains of mu- and delta-opioid receptors and an additional address subsite on the delta-receptor.

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) are the first naturally occurring peptides highly potent for and almost specific to the mu- and delta-opioid receptors, respectively. The amino-terminal domains Tyr-D-X-Phe (where X is either Ala or Met) of these peptides behave as selective and potent mu-receptor ligands. Routing of Tyr-D-X-Phe to the delta- or the mu- receptor is associated with the presence or the absence at the C-terminus of an additional hydrophobic and negatively charged tetrapeptide by-passing the mu-addressing ability of the amino-terminal moiety. A study of 20 Tyr-D-X-Phe-Y-NH2 analogs with substitution of X and Y by neutral, hydrophobic, aromatic amino acids as well as by charged amino acid residues shows that tetrapeptides maintain high binding affinity and selectivity for the mu-opioid receptor. Although residue in position 4 serves a delta-address function, the tripeptide motif at the C-terminus of dermenkephalin and deltorphin I are critical components for high selectivity at delta-opioid receptor. Results demonstrate that mu- and delta-opioid receptors share topologically equivalent ligand-binding domains, or ligand-binding sequences similarities, that recognized Tyr-D-X-Phe as a consensus message-binding sequence. The delta-receptor additionally contains a unique address subsite at or near the conserved binding domain that accommodates the C-terminal tetrapeptide motif of dermenkephalin and deltorphin I.

Amino Acid Sequence↗

PubMed1991

Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin.

A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.

Amino Acid Sequence↗