Loretta Mancinelli

Molecular models of small phosphorylated chromatin peptides. Structure-function relationship and regulatory activity on in vitro transcription and on cell growth and differentiation

We previously reported the isolation of low molecular weight phosphorylated peptides from the chromatin of several tissues. The chromatin peptides show a regulatory activity on DNA in vitro transcription and on cell growth and differentiation. In this paper, we report a molecular model of the native peptides designed according to the structural information obtained by means of biochemical and mass spectrometry analysis: pyroGlu-Ala-Gly-Glu-Asp-Ser(P)-Asp-Glu-Glu-Asn. This or very similar sequences are present in many transcription factors; on the basis of the structural model we presented and of related protein sequences, we have synthesized the peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn. This peptide affects transcription rate in reconstituted systems in vitro and in isolated nuclei; moreover, it inhibits the growth of HL60 cells with a parallel stimulus of differentiation.

S-100ab increases Ca2+ release in purified sarcoplasmic reticulum vesicles of frog skeletal muscle

SummaryThe S-100ab protein, a mixed isoform member of the S-100 family, stimulates Ca2+-induced Ca2+-release from sarcoplasmic reticulum vesicles purified from frog skeletal muscle cells. The effects of S-100ab appear to be specific and result from its peculiar characteristics rather than the fact that it is a calcium-binding protein. Moreover, the addition of S-100ab to the solution completely abolished the inhibition provoked when Ruthenium Red was added alone. Experiments that added labelled Ryanodine with and without S-100 indicated that the protein diminished the affinity of the alkaloid at its receptor site.

Molecular Models of Acidic Peptides from Pea Bud Chromatin and Seminal Plasma. Divalent Cations-Mediated Interaction with DNA

Abstract Small acidic peptides have been isolated from biological fluids (blood and seminal plasma) and from chromatin of several tissues. Their biological activity is related to the control of cell growth and gene expression. This work is an approach to the study of peptide structure-function relationship. Purified fractions from seminal plasma and pea bud chromatin were subjected to fast ion bombardment mass spectrometry. The results obtained were analyzed according to biochemical characteristics of the peptides studied and some possible molecular models have been designed. Two of the proposed sequences were synthesized and their biological activity assayed in cells and cell-free systems. The results demonstrate that the synthetic peptides are able to bind to DNA in the presence of divalent cations (Mg2+, Fe2+, Cu2+) with consequent inhibition of DNA transcription.

A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells.

BackgroundDeproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression.ResultsWe report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression.ConclusionThe data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

Aging Reversibility: from Thymus Graft to Vegetable Extract Treatment – Application to Cure an Age-associated Pathology

Neonatal thymus graft and thymus calf extract (TME) in vivo treatment exert similar corrective actions on different mouse age-related alterations. The aim of the present paper is to investigate whether a vegetal extract, wheat sprout extract (WESPRE), could mimic the thymus action on recovering age-related alterations and if this extract can cure an age-associated pathology, the cataract in dogs. Present experiments were carried out by using WESPRE and TME in vivo in old mice to check their ability to recover the altered DNA synthesis in hepatocyte primary cultures. Old mice treated with WESPRE and TME showed a recovery of hepatocyte DNA synthesis levels when compared with the old untreated ones. The increase of DNA and protein contents observed in aged animals is reduced by WESPRE treatments to levels observed in young mice hepatocytes. We measured also WESPRE phosphorylation activity by endogenous kinase: it was from 10 to 40 times higher with respect to wheat seeds. Old dogs were orally treated for a month and the lens opacity analysed before and after the treatment. Results showed a reduction from 25 to 40% of lens opacity. The efficacy of wheat sprouts in the recovery of age-related alterations and in treating age-associated pathologies could be due to the contemporary presence of small regulatory acid peptides, a remarkable level of highly energetic phosphoric radicals and antioxidant molecules, peculiarities that may be, to some extent, related to the aging process regulation.

Mass spectral and electrophoretic characterization of acidic peptides bound to chromatin of Pea Bud

Structural features of a class of chromatin peptides are studied in the aim of understanding their mechanism of action. They have been reported as a family of small acidic peptides that can affect cell proliferation and RNA transcription. Mass spectrometry analysis has suggested some molecular models of possible sequences that might be present in this group of peptides. These sequences have been synthesised and their chromatographic and electrophoretic behaviour is compared with that obtained from peptides extracted from pea bud chromatin. In this way electric charge and hydrophilic properties of the native peptides are evaluated. On the basis of these data and those obtained from further mass spectrum analysis new models for native peptides are proposed.

The CM2 and CM3 types of α-amylase inhibitor are associated with Triticum aestivum seed chromatin

Abstract Treating the crude chromatin fraction of wheat seed ( Triticum aestivum L.) with alkali and methanol solubilizes several small phosphorylated peptides and a protein fraction, which is almost pure in native and SDS-PAGE analysis. The protein material is composed of two polypeptides, which can be easily separated in PAGE in the presence of 8 M urea, 1 mM 2-mercaptoethanol, 5% (v/v) acetic acid (pH 4.5) and 0.1 (%) v/v Triton X-100. N-terminal sequence analysis revealed that the two polypeptides have sequences that are identical to that of the secreted form of the so-called wheat seed CM2 and CM3 proteins, respectively, belonging to the α-amylase inhibitor superfamily. Their inhibitory activity has been determined in vitro using human salivary amylase and bovine pancreatic trypsin. Immunoblotting experiments, carried out with an antiserum raised in rabbit and which recognizes both CM2 and CM3 proteins, indicate that the protein is mainly expressed in seeds rather than in roots and coleoptiles. In addition, western blotting analysis of protein extracted from highly purified wheat seed chromatin preparation revealed immunoreactivity at the level of the CM2/CM3 protein bands. The possible function of these proteins at the chromatin level has been investigated using gel shift assay and the effect on RNA transcription in vitro. The results indicate that the CM2/CM3 protein binds DNA and inhibits in vitro RNA transcription, suggesting that the protein has a role in the control of the chromatin activity.

Inhibitory effects of agmatine on monoamine oxidase (MAO) activity: Reconciling the discrepancies

Abstract Agmatine has been functionally characterized as an important hormone and co-neurotransmitter in mammals. Given its ability in binding Imidazoline sites, a regolatory site of monoaminoxydase, it has been suggested to be involved in many neurological aspects. However, its inhibitory effect on this enzyme still remains an unanswered question. This present study is aimed to asses whether different experimental conditions could affect the agmatine action on monoaminoxydase activity. We demonstrate that the monoaminoxydase inhibition by agmatine is obtained under alkaline conditions and a long time of incubation. No inhibitiory action was found for shorter times of reaction at elevated pH, or at neutral condition and long time of incubation. No inhibition was also detected by substituting the monoamineoxydase substrate tyramine with kynuramine, however, while in these conditions a remarkable inhibition was shown by two aminoxydase inhibitors tranylcypromine and idazoxan. Herein, we discuss a mechanism model and the functional consequences of agmatine action on monoaminoxydase.