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D Picone

Publications and source records attributed to D Picone.

17 recordsLinked to original sources
PubMed1992

Conformational analysis of an opioid peptide in solvent media that mimic cytoplasm viscosity.

Many neuropeptides exert their action between the presynaptic vesicles and postsynaptic transmembrane receptors, crossing different layers of specialized cytoplasm. Biomimetic media usually employed to study bioactive peptides do not reproduce the physico chemical environment of cytoplasm--in particular, the high viscosity of this biological fluid. Here we describe a conformational study of a delta-selective opioid peptide, deltorphin I, at variable temperatures in several biocompatible media characterized by varying values of viscosity and dielectric constant. It was found that only viscosity, among these parameters, induces ordered conformations; that is, it acts as a conformational sieve. This finding suggests that the high viscosity of the intersynaptic fluid contributes, in addition to the membrane catalysis proposed by Schwyzer, in overcoming the so-called entropic barrier to the transition state of peptide-receptor interaction by selecting ordered conformations prior to receptor interaction. The folded conformer found in the 80:20 (v:v) DMSOd6/H2O cryoprotective mixture at 265 K has a shape consistent with those of rigid nonpeptidic opiates.

Amino Acid Sequence↗
PubMed1991

New insights on mu/delta selectivity of opioid peptides: conformational analysis of deltorphin analogues.

The message domain of dermorphin (Tyr-D-Ala-Phe), a natural mu-opioid heptapeptide, has long been considered the main cause of the high mu selectivity of this peptide and of its analogues. The recent discovery, in the skin of Phyllomedusa sauvagei (i.e., the same natural source of dermorphin) and of Phyllomedusa bicolor of deltorphins, challenges this belief. Deltorphins, in fact, are three heptapeptides characterized by a message domain typical of mu-selective peptides, but endowed of an extremely high delta selectivity, the highest of all natural opioid peptides. A conformational analysis of dermorphin and deltorphins, based on nmr studies in DMSO and cryoprotective mixtures and internal energy calculations, showed that the enormous differences in receptor selectivity can be interpreted on the basis of receptor models for mu and delta opioids that recognize the same beta-turn in the N-terminal part, but discriminate for the conformation and polarity of the C-terminal part. Here we present the synthesis, biological activity, and conformational analysis in solution of three deltorphin analogues with very similar constitution, but with different net charge, different location of negative residues, or even without negative residues, which confirm these hypotheses and show that His4 can play a specific structural role.

Amino Acid Sequence↗
PubMed1990

Conformational preferences of [Leu5]enkephalin in biomimetic media. Investigation by 1H NMR.

The conformation of [Leu5]enkephalin has been studied by 1H-NMR spectroscopy in media more like the actual environment in which the agonist-receptor interaction takes place than water, i.e. in three cryoprotective mixtures (dimethylformamide/water, methanol/water and ethylene glycol/water), in aqueous SDS and in two neat solvents, dimethylformamide and acetonitrile, whose dielectric constants (36.7 and 37.5) are intermediate between that of water and that of the lipid phase. In all cases examined, contrary to the studies in water or dimethylsulfoxide, we were able to detect numerous nuclear Overhauser effects, indicating that the media employed favour well-defined structures and/or reduce the internal motions of the peptide. Data from both organic solvents and cryoprotective mixtures suggest a 4----1 beta turn as the most probable structure of [Leu5]enkephalin in solution, whereas in SDS/H2O micelles the structural picture appears completely different, suggesting the presence of a 5----2 beta turn. The existence of two different preferred conformations of enkephalins may possibly be related to their ability to be effective towards both mu and delta opioid receptors.

Amino Acid Sequence↗
PubMed1990

New features of the delta opioid receptor: conformational properties of deltorphin I analogues.

Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.

Amino Acid Sequence↗
PubMed1989

Conformational properties of deltorphin: new features of the delta-opioid receptor.

Deltorphin is an opioid peptide with the sequence H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the delta-opioid receptor and the similarity of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H-NMR study in two different solvent systems showed that the conformational preferences of the N-terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.

Amino Acid Sequence↗
PubMed1989

Bioactive conformation of linear peptides in solution: an elusive goal?

Bioactive peptides of natural origin have, in general, short linear sequences, and are characterized by a large conformational flexibility. It is very difficult to study their conformation in solution since they exist, almost invariably, as a complex mixture of numerous conformers, most of which are extended. The so-called bioactive conformation may be one of them, although the solvents used in solution studies often have properties drastically different from those of the biological system in which the peptide acts. There is, however, no simple way of identifying the bioactive conformation amid the many existing conformers. It is possible to approach a solution to this problem using two distinct strategies: (a) Limiting the conformational freedom of the peptide, e.g., by increasing the viscosity of the solution and decreasing the temperature, in the assumption that the bioactive conformation is, even slightly, more stable than the others. (b) Trying to mimic in solution the physicochemical features of the more reliable receptor models. These two approaches will be illustrated with examples taken mainly from opioid peptides.

Amino Acid Sequence↗
PubMed1989

Conformational analysis of peptide T and of its C-pentapeptide fragment.

The synthetic peptide of sequence H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, termed peptide T, a competitor of the Human Immunodeficiency Virus in the binding to human T cells, and its C-terminal pentapeptide fragment, were studied by 1H-nmr in DMSO solution to determine conformational preferences. The observation of nuclear Overhauser enhancements (NOEs) for both peptides, and unusual finding for small linear peptides, allowed complete sequence-specific resonance assignments. Long-range NOEs, ring-current shifts, and the very small temperature coefficient of the Thr8 NH chemical shift suggest, for the zwitterionic form of peptide T, the presence in solution of a beta-turn involving Thr5, Asn6, Tyr7 and Thr8. This conformational feature is consistent with previous structure-activity relationship studies indicating the invariance of the same residues in several potent pentapeptide analogues. The studied pentapeptide fragment, although less structured, shows some tendency to fold even in a polar solvent such as DMSO. Preliminary chemotaxis data on some pentapeptide analogues are consistent with our structural model.

HIV↗
PubMed1989

Dissociation and reconstitution of bovine seminal RNAase: construction of a hyperactive hybrid dimer.

The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we described the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.

Amino Acids↗
PubMed1988

A 500 MHz study of peptide T in a DMSO solution.

Peptide T, an octapeptide of sequence ASTTTNYT that binds to human T cells, was studied as a zwitterion in DMSOd6 solution by means of proton NMR spectroscopy at 500 MHz. The unusual dispersion of the resonances of residues of the same type (T) makes it possible to assign all resonances to specific residues by means of several 2D techniques. The non-random nature of the conformation is substantiated by the observation of sequential nuclear Overhauser enhancements (NOEs). The low value of the temperature coefficient of the chemical shift of the NH of T8 and a diagnostic NOE between the NHs of T7 and T8 hint that a beta-turn including T5, N6, Y7 and T8 is a prominent conformational feature in solution. The ring current high field shifts of the methyl group and of the NH of T8 are consistent with an interaction with the side-chain of Y7, favoured by the beta-turn.

Dimethyl Sulfoxide↗
PubMed1986

Biochemical characterization of the monoclonal antibody-defined ovarian carcinoma-associated antigen SGA.

The molecular nature of SGA, the ovarian-carcinoma-associated antigen defined by the MAb OM-1, has been determined. The cell-surface form of the SGA molecule is a glycoprotein with p1 less than 4.2, which on PAGE analysis has an apparent MW of approximately 360 kDa. This was the only OM-1-reactive species found on the cell surface. The apparent MW was unaffected by reducing conditions. The predominant cytoplasmic form of SGA is a non-glycosylated 170-kDa molecule with p1 6.5. Pulse-chase experiments were complicated by the extremely slow rate of SGA synthesis. However, the data indicate that the SGA molecule is synthesized as a 190-kDa protein, cleaved to yield a 170-kDa non-glycosylated intracellular form which is slowly glycosylated to the 360-kDa cell-surface species. Western blotting experiments revealed the presence of the 360-kDa glycosylated molecule in human ovarian cell culture supernatants.

Antibodies, Monoclonal↗
PubMed1979

[Counterimmunoelectrophoresis on cellulose acetate membrane with a commercial lyophilized antigen in diagnosis of human hydatidosis].

A very simplified method of crossed over electrophoresis (CIEP) was employed with a lyophilized commercially produced antigen (previously submitted to several freezing and thawing) and cellulose acetate membrane, for the diagnosis of human hydatidosis. Results are as follows: active hydatidosis (surgically confirmed): number of sera tested 35; positive 32 (91%). Sera of patients with parasitic and non parasitic illnesses (especially malignancies of liver and lung) did not show any precipitin lines. The pattern of immunoprecipitation is characteristically in the form of an "upper lip" or a thick and undulating "streak". The very thin arcs of precipitation which are occurring in some control sera, are to be considered negative. These results indicate that with regard to the rule of the "three S" (specificity, sensitivity and simplicity) the method appears sufficiently satisfactory.

Antibody Formation↗
PubMed1978

[Counterimmunoelectrophoresis in the diagnosis of visceral leishmaniasis].

Technique of counter current immunoelectrophoresis (C.I.E.P.) was employed for the diagnosis of V.L. using an antigen grossly extracted (by means of repeated freezing and thawing) from culture of Leishmania. 6 lots of antigen were prepared - in various time - at the same way. Positive results are obtained in V.L. from 83.3 to 94% (according to various lots of antigens). Few false positivity (from 1.4 to 8.8%) are obtained in sera from patients with other diseases (especially) cirrhosis and blood disorders. No positivity in controls (blood donors). The reproducibility of results appear satisfactory. Our results suggested that C.I.E.P., rapid and less sophisticated test, can be applied for the diagnosis of V.L. But false positivity and false negativity limit the value of these test.

Antibodies↗
PubMed1976

Clearance rate, half-life, and secretory potency of human gastrin-17-I in different species.

The clearance rates of synthetic human gastrin-17-I were measured in man, dog, and cat. Half-life of disappearance and acid secretory potency (D50) were also measured in man and dog. The clearance rates in dog and cat were, respectively, 3 and 8 times more than in man. Accordingly, the half-life of gastrin-17 in the dog (3.5 min) was 3 times shorter than in man (9.5 to 10.5 min). The D50 for acid secretion was proportional to the clearance rate and yielded approximately similar increments of serum gastrin, indicating an equal sensitivty to gastrin-17 at cellular level in the three species. An inverse allometric relation between clearance rate and body weight was consistent with the known greater efficiency of metabolic and eliminatory processes in species of small size. Recent studies of the disposal of other gastrointestinal hormones indicate that the concepts developed theoretically for secretory stimulants and confirmed experimentally for gastrin-17 may have wider applicability.

Animals↗
PubMedDate not supplied

Solution structure of deltorphin I at 265 K: a quantitative NMR study.

Deltorphin I, a delta-selective opioid peptide, has been studied in a DMSOd6/H2O cryoprotective mixture by two-dimensional (2D) NMR spectroscopy in the temperature range 260 K to 305 K. The high viscosity of the solvent at low temperature mimics a distinctive physico-chemical feature of cytoplasm and allows the measurement of a NOESY spectrum rich in intra- and inter-residue effects. Backbone NOEs at 265 K can be calculated with good accuracy in terms of only two limiting conformers: one folded, with a mole fraction of 0.30, and another extended with a mole fraction of 0.70. This calculation is still a rough approximation of the complex conformational equilibria existing in solution but, to the best of our knowledge, is the first one for a flexible peptide, and represents an encouraging starting point for a quantitative evaluation of NMR data of small, flexible peptides in solution. The folded conformer consistent with observed NOEs has a shape surprisingly similar to those of unrelated, rigid, delta-selective opiates.

Amino Acid Sequence↗
PubMedDate not supplied

[CIEP with cellogel (cellulose acetate membrane) in the diagnosis of visceral leishmaniosis (author's transl)].

Technique of counter immunoelectrophoresis (CIEP) was employed for the diagnosis of V.L. (human and canine) using strips of cellulose acetate and an antigen grossly extracted (by means of repeated freezing and thawing) from culture of Leishmania. 4 lots of antigen was prepared, in various time, at the same way. Positive results were obtained in V.L. from 81 to 90% (according to various lots of antigen). False positivities (from 1 to 4.5%) occur in patient with other diseases (especially cirrhosis and blood disorders). None positivity in controls (blood donors). Present results and those obtained with same technique in agar, suggested the validity of method and encouraged the production of antigen in a "kit" form for use in the field.

Animals↗