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

    Publication Date: 11/09/1990, on European journal of biochemistry
    by Picone D, D'Ursi A, Motta A, Tancredi T, Temussi PA

    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.

  • Regenerated EDL muscle of rats requires innervation to maintain AChE molecular forms.

    Publication Date: 01/08/1990, on Muscle & nerve
    by Melone MA, De Lucia D, Fratta M, Cotrufo R
    DOI: 10.1002/mus.880130809

    Extensores digitorum longi of rats, infarcted and denervated by different surgical procedures, were used to analyze by biochemical and cytochemical methods the acetylcholinesterase (AChE) changes during muscle degeneration, regeneration, and early or delayed reinnervation. Biochemical tests showed that the regenerating muscle produces globular AChE forms (36% of controls) and small amounts of A12 (16S) asymmetric form (5% of controls); at the end of the regeneration, innervation and electromechanical function are required for the complete recovery of globular forms, and are absolutely critical to prevent A12 (16S) disappearance. Cytochemical observations showed that, unlike nicotinic receptor, AChE deposited at the neuromuscular junction before ischemic necrosis is protected from breakdown, as is the basal lamina of muscle fibers. Taken together, these observations contribute to the understanding of the factors that play a critical role in muscle repair and are, therefore, of clinical relevance.

  • Autoinduction of estrogen receptor is associated with FOSP-1 mRNA induction by estrogen in primary cultures of Xenopus oviduct cells.

    Publication Date: 09/07/1990, on Molecular and cellular endocrinology
    by Varriale B, Tata JR

    The number of nuclear and cytosolic estrogen receptors (ER) per cell and the steady-state levels of the mRNA encoding a tissue-specific, estrogen-inducible protein (FOSP-1) were measured as a function of time following the addition of estradiol-17 beta (E2) to primary cultures of Xenopus oviduct cells. After a lag period of about 12 h, 10(-9) to 10(-7) M E2 caused a 10 to 15-fold increase in FOSP-1 mRNA by 60 h, whereas it was only 2-fold with 10(-7) M progesterone. Under the same conditions, E2 doubled its own total receptor content within the first 12 h, reaching a 4-fold increase in nuclear ER by 48 h. Cycloheximide treatment in the presence of 10(-7) M estradiol reduced the functional ER content by 75.90%. Treatment with the anti-estrogen ICI 164,384 of oviduct cells in which FOSP-1 mRNA was pre-induced to high levels with the hormones caused a drastic reduction in nuclear ER and a total loss of FOSP-1 mRNA in 72 h. The close correlation between the kinetics of autoinduction of ER and the induction of FOSP-1 mRNA, as was shown earlier for vitellogenin mRNA in hepatocytes (Perlman et al. (1984) Mol. Cell. Endocrinol. 38, 151-161), strongly suggests that Xenopus egg protein gene activation by estrogen requires the up-regulation of its own receptor by the hormone.

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

    Publication Date: 15/06/1990, on Biochemical and biophysical research communications
    by Balboni G, Marastoni M, Picone D, Salvadori S, Tancredi T, Temussi PA, Tomatis R

    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.

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

    Publication Date: 01/12/1989, on Journal of protein chemistry
    by Tamburrini M, Piccoli R, Picone D, Di Donato A, D'Alessio G

    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.

  • Histopathological heterogeneity and cytopathological similarity of findings in different muscles of two brothers affected by rigid spine syndrome.

    Publication Date: 01/12/1989, on Journal of the neurological sciences
    by Di Iorio G, Lus G, Cutillo C, Cecio A, Cotrufo R

    The pathological changes in muscles biopsied from 2 brothers with rigid spine syndrome are reported. The findings ranged from marked fascicular atrophy and fibrosis to hypotrophy of small groups of fibres and vacuolation in most fibres. The presence of vacuoles and deposits of accumulated material seemed to be common to all the biopsies. These findings, compared with those reported in the literature, confirmed the histopathological heterogeneity of this syndrome but proposed also the hypothesis that similar elementary lesions of muscle fibres can account for the initiation of the pathological process, developing asynchronously in different muscles because of their different activity.

  • Thyrotropin stimulates transcription from the ferritin heavy chain promoter.

    Publication Date: 30/11/1989, on Biochemical and biophysical research communications
    by Colucci-D'Amato LG, Ursini MV, Colletta G, Cirafici A, de Francisis V

    Thyrotropin (TSH) is the primary hormone regulating the activity of the thyroid gland. We have recently shown that TSH stimulates H-ferritin mRNA levels in rat thyroid. Ferritin plays a key role in determining the intracellular fate of iron. The induction of ferritin synthesis by iron in liver is regulated both at transcriptional and translational levels. Here we present evidence that the mechanisms by which TSH regulates the mRNA levels are mediated by a diffusible product acting in trans on its own promoter. In fact, the H-ferritin promoter mediates increased CAT activity in response to hormone induction. Our results identify transcription as an important regulatory step of TSH action. They suggest that TSH induces expression of the ferritin gene, and that continuous protein synthesis is required to maintain basal ferritin gene expression in the absence of hormone.

  • Memory encoding processes in mild and severe dementia. Preliminary data.

    Publication Date: 01/10/1989, on Functional neurology
    by Chiacchio L, Trojano L, Orsini A, Grossi D

    Memory coding processes can be divided according to their attentional requirement into "automatic" and "effortful". The aim of current research was to explore both automatic and effortful processes in early and late stages of Alzheimer type dementia. Patients were classified on the basis of restrictive clinical and neuropsychological criteria, in order to pinpoint possible specific impairments in the two patients' subgroups. The present paper reports preliminary data about 15 mildly and 15 severely demented patients. Statistical analysis showed that both the automatic and the effortful coding processes are impaired at the late but also at the early stages of dementia.

  • On the different roles of the cerebral hemispheres in mental imagery: the "o'Clock Test" in two clinical cases.

    Publication Date: 01/05/1989, on Brain and cognition
    by Grossi D, Modafferi A, Pelosi L, Trojano L

    This paper develops the hypothesis of different roles of the two hemispheres in imaginal processes. A test designated the "o'Clock Test" is proposed, which contains closely confrontable perceptual and imaginal tasks. It enables examination of the abilities both to generate mental images and to explore them in their right and left halves (R + L Condition). This test was used to examine two patients, one severely affected by hemi-inattention resulting from a right posterior lesion, and the other with a selective deficit of imagery due to a left occipital lesion. The former demonstrated left neglect in both perceptive and imaginal capacities, while the latter was able to perform correctly only the perceptive tasks. These results suggest that the right hemisphere has functions of organization and spatial exploration at both perceptive and imaginal levels, and that the left hemisphere's role is to generate mental images.

  • Normalization of short-chain acylcoenzyme A dehydrogenase after riboflavin treatment in a girl with multiple acylcoenzyme A dehydrogenase-deficient myopathy.

    Publication Date: 01/05/1989, on Annals of neurology
    by DiDonato S, Gellera C, Peluchetti D, Uziel G, Antonelli A, Lus G, Rimoldi M
    DOI: 10.1002/ana.410250510

    A 12-year-old girl was shown to have carnitine-deficient lipid storage myopathy and organic aciduria compatible with multiple acylcoenzyme A (acyl-CoA) dehydrogenase deficiency. In muscle mitochondria, activities of both short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) were 35% of normal. Antibodies against purified SCAD, MCAD, and electron-transfer flavoprotein were used for detection of cross-reacting material (CRM) in the patient's mitochondria. Western blot analysis showed absence of SCAD-CRM, reduced amounts of MCAD-CRM, and normal amounts of electron-transfer flavoprotein-CRM. The patient, who was unresponsive to treatment with oral carnitine, improved dramatically with daily administration of 100 mg oral riboflavin. Increase in muscle bulk and strength and resolution of the organic aciduria were associated with normalization of SCAD activity and "reappearance" of SCAD-CRM. In contrast, both MCAD activity and MCAD-CRM remained lower than normal. These results suggest that in some patients with multiple acyl-CoA dehydrogenase deficiency riboflavin supplementation may be effective in restoring the activity of SCAD, and possibly of other mitochondrial flavin-dependent enzymes.

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

    Publication Date: 24/04/1989, on FEBS letters
    by Temussi PA, Picone D, Tancredi T, Tomatis R, Salvadori S, Marastoni M, Balboni G

    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.

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

    Publication Date: 01/01/1989, on Biopolymers
    by Motta A, Picone D, Temussi PA, Marastoni M, Tomatis R
    DOI: 10.1002/bip.360280142

    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.

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

    Publication Date: 01/01/1989, on Biopolymers
    by Temussi PA, Picone D, Castiglione-Morelli MA, Motta A, Tancredi T
    DOI: 10.1002/bip.360280112

    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.

  • Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate. Detection of the reaction products by fast-atom-bombardment mass spectrometry.

    Publication Date: 15/10/1988, on European journal of biochemistry
    by Galletti P, Ingrosso D, Manna C, Sica F, Capasso S, Pucci P, Marino G

    Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595-602], while very little is known concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11,503-11,511]; (2) the isoaspartyl residue is not recognized unless its alpha-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methyl-esterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.

  • Selective "semantic amnesia" after closed-head injury. A case report.

    Publication Date: 01/09/1988, on Cortex; a journal devoted to the study of the nervous system and behavior
    by Grossi D, Trojano L, Grasso A, Orsini A

    An 18-year-old girl with a left parietal lesion caused by a closed-head injury exhibited a peculiar memory impairment. The patient showed selective "semantic amnesia" (severe loss of the stock of notions acquired prior to the disease), while autobiographic memory and spatial learning ability were preserved. The semantic/episodic distinction is discussed in the light of the patient's performance on neuropsychological tasks.