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P Rovero

Publications and source records attributed to P Rovero.

At least 55 records · Page 3Linked to original sources
PubMed1993

Rapid simultaneous determination of tryptophan and tyrosine in synthetic peptides by derivative spectroscopy.

A method for the simultaneous quantitative determination of tryptophan and tyrosine in synthetic peptides by second-order derivative diode-array spectroscopy is reported. The method does not require hydrolysis of the peptides or a derivatization reaction; the sample is dissolved in 0.1 N NaOH and directly scanned between 262 and 264 nm to detect tyrosine and between 304 and 306 nm to detect tryptophan. From these results the peptide content of the synthetic sample can be easily calculated in a very simple and fast way. The results obtained by the described method with several peptides are compared with those obtained by classical high-performance liquid chromatographic analysis of amino acids after peptide hydrolysis. A very good correlation was found both for the tryptophan/tyrosine molar ratio and the peptide content.

Amino Acid Sequence↗
PubMed1993

HLA class I binding of synthetic nonamer peptides carrying major anchor residue motifs of HLA-B27 (B*2705)-binding peptides.

Eight nonamer peptides that comply with the major anchor residue motifs (the combination of amino acid residues at positions 2 and 9), R - K and R - R, of HLA-B27 (B*2705)-binding peptides were synthesized and tested for their direct binding to HLA class I alpha chains by the HLA class I alpha chain refolding assay previously described. One was a known B27 (B*2705)-binding heat shock protein peptide, HSP89 alpha (201-209), and the other seven were derived from the sequence of wild-type P53, a human tumor suppressor protein. A total of 36 HLA class I allospecificities were tested. HSP89 alpha (201-209) and two P53 peptides, P53 (362-370) and P53 (378-386), all possessing the motif R - K, bound strongly to B27 (B*2705) alpha chains. A weak binding was seen for P53 (272-280) and P53 (334-342), both showing the motif R - R. Most of these B27-binding peptides were found to bind to A3 alpha chains as well. In addition, P53 (173-181) and P53 (334-342), both with the R - R motif, showed substantial binding with A31 alpha chains. All the peptides carrying the motif R - K also showed weak binding with A31 alpha chains. The remaining two peptides, P53 (201-209) and P53 (282-290), with the motif R - R, did not show significant binding with any of the alpha chains tested. This study demonstrates both the specificity of peptide binding to a given HLA allelic product and the occurrence of cross-peptide-binding between the allelic products of different HLA loci.

Amino Acid Sequence↗
PubMed1993

Anchor residue motifs of HLA class-I-binding peptides analyzed by the direct binding of synthetic peptides to HLA class I alpha chains.

The binding characteristics of the primary anchor residue motifs reported for HLA-A2 (A*0201, A*0205) and HLA-B27 (B*2705) alleles were investigated by a direct binding assay of the pertinent synthetic peptides to HLA class I alpha chains derived from a panel of HLA homozygous B-cell lines of various HLA phenotypes, including four A2 subtypes. The assay is based on a serologic detection of the conformational change of HLA class I alpha chains induced by binding to specific peptides in the presence of beta 2m. It is applicable to test a large number of HLA allelic products and synthetic peptides. Assay data confirmed the high allele specificity of the anchor residue motifs tested, but also revealed the intra- and interlocus cross-reactivity of these motifs. In the case of A2 anchor motifs, not only a broad cross-reactivity within the A2 subgroup, but also cross-reactivities with A24, A26, A28, and A29 were observed. With B27 anchor motifs, an interlocus cross-reactivity with A3 and A31 was seen. Several peptides, even though they carried A2 or B27 major anchor residue motifs, failed to bind to the relevant alpha chains, suggesting that the presence of a primary anchor residue motif is necessary for HLA class-I-peptide binding but is not by itself sufficient to guarantee binding.

Amino Acid Sequence↗
PubMed1993

Characterization of the tachykinin neurokinin-2 receptor in the human urinary bladder by means of selective receptor antagonists and peptidase inhibitors.

The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

Adult↗
PubMed1993

Role of C-terminal amidation on the biological activity of neurokinin A derivatives with agonist and antagonist properties.

The mammalian tachykinins, neurokinin A (NKA) and NKA(4-10), along with the tachykinin NK2 receptor-selective antagonist MEN 10,376, were compared to their C-terminal free acid derivatives, NKA-OH, NKA(4-10)-OH and MEN 10,456, respectively, on several in vitro bioassays for NK1, NK2 and NK3 tachykinin receptors. NKA-OH and NKA(4-10)-OH were much weaker agonists than NKA or NKA(4-10) in the endothelium-deprived rabbit pulmonary artery (endowed with NK2A receptors) and in the guinea pig isolated bronchus (endowed with NK2A and NK1 receptors), where they produced submaximal contractile responses, and were inactive in the hamster isolated trachea (endowed with NK2B receptors) and in the rat isolated portal vein (endowed with NK3 receptors). At NK1 receptors of the guinea pig isolated ileum, NKA-OH produced weak agonist responses, whereas NKA(4-10)-OH was ineffective. In sharp contrast, MEN 10,456, while maintaining the same antagonist potency of the parent compound MEN 10,376 in the rabbit pulmonary artery and hamster isolated trachea, developed a clear-cut agonist character in the rat isolated portal vein, guinea pig isolated ileum and guinea pig isolated bronchus. The agonist responses produced by MEN 10,456 (10 microM) were reduced by MEN 10,376 in the guinea pig isolated bronchus and by the NK1 receptor antagonist GR 82,334 in the guinea pig isolated ileum. These results, although indicating the importance of C-terminal amidation for the agonist activity of natural tachykinins, suggest that the C-terminal amide group may not be directly involved in stimulation of the tachykinin receptors, but could induce agonist activity through a conformation effect.

Amides↗
PubMed1992

[125I]His-neurokinin A binds selectively to NK2 receptors of the B-type in rat small intestine smooth muscle membranes.

The high-affinity, reversible binding of [125I]His-neurokinin A (NKA) to rat small intestine smooth muscle membranes was investigated. Endogenous neurokinin agonists, selective neurokinin analogues, both agonist and antagonist, were used to define the selectivity of the binding. Both the endogenous and selective neurokinin analogue agonists displayed orders of potency indicating that [125I]His-NKA was binding to NK2 receptors. The use of recently developed NK2-selective antagonists indicated that the NK2 receptors present in this preparation were similar to those described in hamster trachea preparations (NK2B), and not endothelium-denuded rabbit pulmonary artery (NK2A). The absence of NK2A receptors and the predominance of NK2B was confirmed by blocking experiments using MEN10376 and L659877. Low-affinity binding of NKA was also observed with this preparation, which was not sensitive to the NK2-selective agonist, [beta-Ala8]NKA4-10. This was shown not to be due to the presence of NK1 or NK3 receptors by using selective agonists for NK1 and NK3 to block any such receptors. (No evidence for the presence of these receptors was obtained during these blocking experiments.) Guanylylimidodiphosphate appears to discriminate between the high- and low-affinity binding sites for NKA. It was thus concluded that high-affinity binding of [125I]His-NKA to rat small intestine smooth muscle membranes was selective for NK2B receptors. No evidence was found for the binding of [125I]His-NKA to NK1, NK3 or NK2A receptors.

Animals↗
PubMed1992

Activity of peptide and non-peptide antagonists at peripheral NK1 receptors.

We investigated the affinity of several tachykinin antagonists reportedly selective for NK1 receptors at various tachykinin receptors and NK2 receptors subtypes. The four antagonists tested were: L 668,169, Spantide II, Ac-Thr-DTrp(for)-Phe-NMeBzl (FR 113680) and the novel nonpeptide antagonist (+/-)-CP-96,345. The four antagonists were found to be effective against NK1 receptor-mediated responses in the guinea-pig ileum with the following rank order of potency (pKB values in parentheses): (+/-)-CP-96,345 (8.11) greater than Spantide II (7.08) greater than FR 113680 (6.61) greater than or equal to L 558,169 (6.44). (+/-)-CP-96,345, Spantide II and FR 113680 were distinctly more potent at NK1 receptors than at NK2 receptors (NK2A in the rabbit pulmonary artery, NK2B in the hamster trachea). L 668,169 antagonized neurokinin A-induced contractions in the hamster trachea with an affinity similar (pKB value 6.16) to that found in the guinea-pig ileum for NK1 receptors (pKB value 6.44). All antagonists were inactive at NK3 receptors of the rat portal vein. In a second series of experiments, the affinities of test antagonists for NK1 receptors in the guinea-pig ileum were compared to those for NK1 receptors in the guinea-pig vas deferens, the rabbit jugular vein and the rat urinary bladder. For each antagonist, the affinity measured in the guinea-pig vas deferens and the rabbit jugular vein was comparable to that found in the guinea-pig ileum. In the rat urinary bladder, (+/-)-CP-96,345 was about 100 times less potent in blocking NK1 receptor-mediated contractions than in the guinea-pig ileum.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

N-terminal truncated analogs of men 10376 as tachykinin NK-2 receptor antagonists.

Three N-terminal fragments of the selective tachykinin NK-2 receptor antagonist MEN 10376 (H-Asp-Tyr-DTrp-Val-DTrp-DTrp-Lys-NH2) have been synthesized and tested in several mammalian tissues in order to establish the minimum length of the peptide chain for maintenance of the antagonist activity. Biological activity has been determined on the rabbit pulmonary artery (RPA) and hamster trachea (HT) preparations, chosen as representative of the NK-2A and NK-2B receptor subtypes, respectively, and on the rabbit bronchus (RB), guinea-pig bronchus (GPB), human urinary bladder (HuUB), human ileum (HuI) and human colon (HuC) preparations to verify the previously described NK-2A character of these tissues. The N-terminal tetrapeptide was inactive in the RPA and HT, while the N-terminal hexa- and penta- peptides maintained antagonist activity in all preparation investigated. The selectivity of the latter two peptides confirms that the receptor expressed in RB, GPB, HuUB, HuC and HuI tissues is of the NK-2A type.

Amino Acid Sequence↗
PubMed1992

Affinity of R 396, an NK-2 tachykinin receptor antagonist, for NK-2 receptors in preparations from different species.

We have assessed the affinity of R 396 (Ac. Leu-Asp-Gln-Trp-Phe-Gly NH2) in a number of NK-2 tachykinin receptor bearing-tissues from several species. The cyclic analog of R 396, (MEN 10354) was less potent and selective than the linear hexapeptide at NK-2 tachykinin receptors subtypes in the rabbit pulmonary artery and hamster trachea. The affinity of R 396, as measured by a smooth muscle contraction assay and a radioligand binding assay, was higher (about 10 fold) for NK-2 receptors expressed in hamster tissues (urinary bladder, stomach and trachea) than in rat tissues (urinary bladder, vas deferens, colon and stomach) and a further drop in affinity was observed in bovine tissues (urinary bladder and stomach) or rabbit bronchus. The results are discussed in relation to the proposed existence of NK-2 receptor subtypes and raise the question of the existence of species-related differences as compared to the existence of true receptor subtypes.

Amino Acid Sequence↗
PubMed1992

Interaction of amyloid beta protein (25-35) with tachykinin receptors.

Amyloid beta protein (25-35) failed to significantly interact with tachykinin NK1 (rat forebrain, guinea-pig ileum) NK2 (rabbit pulmonary artery, hamster trachea) or NK-3 (guinea-pig cortex) receptors, as determined by radioligand binding and functional assays. A weak interaction (Ki 14.8 microM) was detected with NK2 receptors in rat small intestine. It appears unlikely that direct interaction with tachykinin receptors may account for the reported ability of amyloid beta protein (25-35) to affect neuronal survival.

Amino Acid Sequence↗
PubMed1992

Structure-activity relationship study of R396, an NK2 tachykinin antagonist selective for the NK2B receptor subtype.

We report on a structure-activity study of R396 (Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2), a linear hexapeptide tachykinin antagonist selective for the putative NK2B receptor subtype. Asp2, Trp4 and the C-terminal glycinamide have been challenged by classical amino acid substitutions with the aim of elucidating the structural requirements responsible for NK2 subtype selectivity. The biological activities indicate that Asp2 has a crucial role for the high affinity of R396 at the NK2B subtype: none of the analogues substituted in position 2 display higher affinity as compared to R396, regardless of the nature of the residue introduced. Trp4 has been replaced by other aromatic residues, again yielding weak antagonist or inactive compounds. Finally, the C-terminal amide appears to be crucial for affinity, the free acid analogue being devoid of biological activity. On the other hand, antagonistic activity is maintained both by the desGly pentapeptide and by the analogue bearing beta Ala in place of Gly in position 6. In conclusion, since the NK2B selectivity pattern was maintained throughout the whole series of R396 replacement analogues, we speculate that the overall conformational features of this family of linear hexapeptides favour the interaction with the NK2B receptor subtype.

Amino Acid Sequence↗
PubMed1992

Heterogeneity of tachykinin NK-2 receptors in rabbit, guinea-pig and human smooth muscles.

Recent evidence indicates that the tachykinin NK-2 receptor is heterogenous (subtypes/species variants) and the existence of NK-2A (or 'non-classical') and NK-2B (or 'classical') forms of the NK-2 receptor in mammalian tissues has been proposed. In this study we have compared the affinities of 7 linear octa- and heptapeptide derivatives of neurokinin A (4-10) and that of two cyclic hexapeptides endowed with selective NK-2 receptor antagonist properties in 5 mammalian smooth muscle preparations previously characterized as expressing the NK-2A receptor subtype (rabbit pulmonary artery and bronchus, guinea-pig bronchus, human ileum and colon) and 2 preparations previously characterized as expressing the NK-2B receptor subtype (rat vas deferens and hamster trachea). The results of this comparative study reinforce the concept of two broad categories of preparations expressing pharmacologically distinguishable forms of the tachykinin NK-2 receptor and suggest the possibility of a further heterogeneity within the previously defined NK-2A receptor subtype.

Amino Acid Sequence↗
PubMed1991

NK2 tachykinin receptors and contraction of circular muscle of the human colon: characterization of the NK2 receptor subtype.

The contractile effect of substance P, neurokinin A, receptor selective agonists for tachykinin receptors and NK2 tachykinin receptor antagonists was investigated in mucosa-free circular strips of the human isolated colon. Neurokinin A and substance P produced concentration-dependent contractions which approached 80-90% of the maximal response to carbachol. Neurokinin A was about 370 times more potent than substance P. The action of neurokinin A and substance P was not modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The NK2 receptor selective agonist, [beta-Ala8]neurokinin A-(4-10) closely mimicked the response to neurokinin A while NK1 and NK3 receptor selective agonists were active only at microM concentrations. The pseudopeptide, MDL 28,564, which is one of the most selective NK2 ligands available, behaved as a full agonist. Responses to [beta-Ala8]neurokinin A were antagonized by NK2 receptor selective antagonists, with the rank order of potency MEN 10,376 greater than L 659,877 much greater than R 396. These data indicate that NK2 tachykinin receptors play a dominant role in determining the contraction of the circular muscle of the human colon to peptides of this family. The NK2 receptor subtype responsible for this effect belongs to the same subtype (NK2A) previously identified in the rabbit pulmonary artery and guinea-pig bronchi.

Aged↗
PubMed1991

Conformation-activity relationship of tachykinin neurokinin A (4-10) and of some [Xaa8] analogues.

NKA (4-10), the C-terminal heptapeptide fragment (Asp-Ser-Phe-Val-Gly-Leu-Met-NH2) of tachykinin NKA, is more active than the parent native compound in the interaction with the NK-2 receptor. Substitution of Gly8 with the more flexible residue beta-Ala8 increases its selectivity with respect to other two known receptors (NK-1 and NK-3), whereas substitution with either D-Ala8 or GABA8 deprives the peptide of its biological activity. These findings can be interpreted by a conformational analysis based on NMR studies in DMSO-d6 and in a DMSO-d6/H2O cryoprotective mixture combined with internal energy calculations. NKA(4-10) is characterized by a structure containing a type I beta-turn extending from Ser5 to Gly8, followed by a gamma-turn centered on Gly8, whereas for [beta-Ala8]NKA(4-10) is possible to suggest a type I beta-turn extending from Ser5 to beta-Ala8, followed by a C8 turn comprising beta-Ala8 and Leu9 and by another beta-turn extending from beta-Ala8 to the terminal NH2. The preferred conformation of [beta-Ala8]NKA(4-10) is not compatible with models for NK-1 and NK-3 agonists proposed on the basis of rigid peptide agonists [Levian-Teitelbaum et al. (1989) Biopolymers 28, 51-64; Sumner & Ferretti (1989) FEBS Lett. 253, 117-120]. The preferred solution conformation of [beta-Ala8]NKA(4-10) may thus be considered as a likely bioactive conformation for NK-2 selective peptides.

Amino Acid Sequence↗
PubMed1991

Intracerebroventricular administration of endothelins: effects on the supraspinal micturition reflex and blood pressure in the anaesthetized rat.

Intracerebroventricular (i.c.v.) administration of endothelin-1 (ET-1, 10 and 100 pmol/rat) or endothelin-3 (ET-3, 100 pmol/rat) inhibited the supraspinal micturition reflex (SMR) in urethane-anesthetized rats. Guanethidine pretreatment, vagotomy or forced ventilation did not modify the effect of i.c.v. ET-1 on the SMR. Intravenous (i.v.) administration of ET-1 (350 pmol/rat) also blocked the SMR with a delay of about 8 min. Guanethidine pretreatment revealed a stimulatory effect of i.v. ET-1 on the frequency of the SMR and the late inhibition was also reduced. In the isolated rat urinary bladder ET-1, ET-3 and ET-(16-21) induced a dose-related contraction and a small enhancement of the neurogenic contraction caused by electrical field stimulation. Inhibition of bladder motility by i.c.v. administration of ET-1 or ET-3 was paralleled by an increase in blood pressure. Both ET-1 and ET-3, at the same doses which evoked these autonomic effects, also killed some animals. The ET-1-induced increase in blood pressure was not prevented by guanethidine pretreatment or by spinal cord transection. Guanethidine pretreatment also did not modify the pressor response induced by i.v. ET-1. Bilateral cervical vagotomy enhanced the pressor response to i.c.v. ET-1. Forced ventilation prevented the death of some animals following i.c.v. administration of ET-1.

Animals↗
PubMed1991

Tachykinin receptors in the guinea-pig isolated bronchi.

The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery.

Amino Acid Sequence↗