Bimodal effect of D-aspartate on brain aging processes: insights from animal models.

Nowadays it is widely recognized that D-amino acids are present in bacteria as well as in eukaryotes, including mammals. In particular, free D-serine and D-aspartate are found in the brain of mammals. Notably, D-aspartate occurs at substantial levels in the embryo brain to then consistently decrease at post-natal phases. Temporal regulation of D-aspartate content depends on the post-natal onset of D-aspartate oxidase expression, the only known enzyme able to catabolize this D-amino acid. Pharmacological evidence indicates that D-aspartate binds and activates NMDA receptors (NMDARs). To decipher the physiological function of D-aspartate in mammals, in the last years, genetic and pharmacological mouse models with abnormally higher levels of this D-amino acid have been generated. Overall, these animal models have pointed out a significant neuromodulatory role for D-aspartate in the regulation of NMDAR-dependent functions. Indeed, increased content of D-aspartate are able to increase hippocampal NMDAR-dependent long-term potentiation (LTP) and spatial memory of adult mice. However, if exposure to elevated levels of D-Asp lasts for the entire lifetime of mice, enhancement of synaptic plasticity turns into a dramatic worsening, thus triggering an acceleration of the NMDAR-dependent aging processes in the hippocampus. Nonetheless, administration of D-Asp to old mice can restore the physiological age-related decay of hippocampal NMDA-related LTP. Besides its effect on hippocampus-dependent processes in mouse models, different points of evidence are indicating, today, a potential role for D-Asp in neurologic and psychiatric disorders associated with aberrant signalling of NMDARs.

Wheat sprout extract induces changes on 20S proteasomes functionality.

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols. The antioxidant properties of wheat sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells. The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin. The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. beta-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12-O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins.

Binding citrate/DNA in presence of divalent cations. Potential mimicry of acidic peptides/DNA interactions.

Citric acid whose structure is comparable to that of small acidic peptides, can bind to DNA in the presence of divalent cations (Cu2+, Fe2+, Zn2+, Mg2+). Citrate-DNA interaction occurs also in a cell homogenate and in this experimental model too requires the presence of natural divalent cations. In fact the addition of 2 mM EDTA to cell homogenate strongly decreases the DNA-citrate binding. The results demonstrate that divalent cations can act as bridges between two acidic molecules and that citric acid can mimic the structure of acidic peptides.

Competition between citrate and heptapeptide DDSDEEN binding to DNA in presence of divalent cations.

The binding of citrate and acidic peptide DDSDEEN with DNA in the presence of divalent cations is compared. Citric acid shows a higher number of binding sites on the DNA compared to the peptide; this is probably due to the bigger sitric hindrance of the peptide compared to the citric acid for the binding in the DNA grooves. Moreover. DNA preincubated with saturating amounts of citric acid is not available for the binding with successively added peptide. Therefore the peptide and citrate binding sites to some extent overlap.

Small acidic peptides and peptidomimetic molecules: cell growth and differentiation.

Small phosphorylated chromatin peptides exert a homeostatic regulation on gene expression which causes a strong inhibition of RNA synthesis and growth of neoplastic and fast-growing cells and a remarkable activation of metabolic pathways slowed down in ageing. By biochemical and mass spectrometry analysis, some molecular models of these peptides have been designed and synthesised. Recent studies show that it is possible to find peptidomimetic structures, such as citric acid, able to reproduce the antiproliferative effect. The mechanism of action has been investigated and partially clarified.

Molecular models of acidic peptides from pea bud chromatin and seminal plasma. Divalent cations-mediated interaction with DNA.

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.

Dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn inhibits the proliferation rate of HL-60 cells.

Small acidic phosphorylated chromatin peptides show regulatory activity on gene expression. The peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn, synthesized on the basis of structural and biochemical studies, shows functional properties in vitro (phosphorylation by casein kinase II, control of DNA transcription by RNA polymerase II, inhibition of proliferation and promotion of differentiation in some cell lines) very similar to those of native chromatin peptides. In this report we show that the dansylated octapeptide Dns-Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn remarkably inhibits cell growth of the HL-60 cell line. The biological effect of the peptide seems to be considerably higher than that shown by the nondansylated peptide, and it cannot be attributed to a toxic effect of the Dns group. The measurement of uptake of 3H-labelled Glu-Asp-Asp-Ser-Asp-Glu-Glu-Asn demonstrates that it is unable to pass through the HL-60 cell membrane. It is our considered opinion that the addition of hydrophobic groups to the peptide N-terminus should increase the biological activity by improving its transport through the cellular membrane.

Synthetic octapeptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn promotes differentiation in promyelocytic HL-60 cell line.

The activity of the octapeptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn in the control of cell growth and differentiation of human myeloblastic leukemia cells HL-60 is reported. Treatment with peptide slightly slows down the rate of cellular proliferation and this effect becomes more evident in cells grown for several weeks in the presence of the effector. An enhanced effect (40-50% inhibition respect to the control) is found in reversibly permeabilized cells and after 1% DMSO is added to the medium. Moreover the presence of peptide markedly increases the percentage of cells differentiated by DMSO and RA. The effect in DMSO-induced cells is more evident than that observed in RA-induced cells. This in agreement with our hypothesis that DMSO facilitates the peptide entry and its effect is due to an intracellular action.

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.

Synthetic octapeptide pyroGLU-ASP-ASP-SER-ASP-GLU-GLU-ASN controls DNA transcription in vitro by RNA polymerase II.

The effect of the synthetic octapeptide pyroGLU-ASP-ASP-SER-ASP-GLU-GLU-ASN (phosphorylated by casein kinase II, CKII) on DNA transcription by RNA polymerase II has been studied. The peptide contains the acidic carboxy-terminus heptapeptide of the largest subunit of RNA polymerase II, which has been demonstrated to be a phosphorylation site for CKII. The aim of this work is to obtain some insights about the possible role of this domain in RNA polymerase II activity and DNA binding. Results demonstrated that the phosphorylated octapeptide causes strong inhibition of transcription of calf thymus DNA or pSVL SV40 plasmid DNA by RNA polymerase II, when used at concentrations between 0.4-4 micrograms/ml.

Phosphorylation of synthetic acidic peptides by casein kinase II: evidence for competition with phosphorylation of proteins involved in transcription.

Phosphorylation of several synthetic acidic peptides by biochemically isolated casein kinase II (CKII) and by cellular and nuclear extracts containing CKII-like activity has been investigated. Especially the synthetic peptide pyroGlu-Asp-Asp-Ser-Asp-Glu-Glu-Asn comprising the carboxy-terminal acidic hepta-peptide of the largest subunit of RNA polymerase II was found to serve as an excellent substrate for purified CKII. Moreover, this peptide reduces the rate of 'in vitro' ATP-dependent stimulation of DNA transcription induced by the proteins in the extracts. Since the peptide itself is also significantly phosphorylated in such assays, it is supposed that it serves as a competitive substrate for the phosphorylation of proteins in the extracts whose phosphorylation seems to be a prerequisite for their activity in the transcription process. This points to the involvement of CKII and substrate(s) of CKII in the process of transcription.

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

The S-100ab protein, a mixed isoform member of the S-100 family, stimulates Ca(2+)-induced Ca(2+)-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.

S-100a0 protein stimulates Ca2+-induced Ca2+ release from isolated sarcoplasmic reticulum vesicles.

S-100a0 protein, the alpha alpha-isoform of the S-100 family, stimulates Ca2+-induced Ca2+ release from terminal cisternae isolated from rat skeletal muscle cells. The stimulatory effect of S-100a0 is maximal at approximately 5 microM S-100a0 and half maximal at approximately 0.1 microM S-100a0, at 1.8 microM free Ca2+ in the presence of 5 mM Mg2+ plus 0.1 M KCl. The effect of the protein on Ca2+-induced Ca2+ release is completely inhibited by the calcium release blocker, ruthenium red.

Functional aspects of calcium transport in sarcoplasmic reticulum vesicles derived from frog skeletal muscle treated with saponin.

To study the physiological aspects of the excitation-contraction cycle, saponin (10-100 micrograms ml-1) was used as a skinning agent on muscle and sarcotubular vesicles derived from fast muscles (sartorius and tibialis anterior) of Rana esculenta. The vesicles showed similar Ca2+-ATPase activity and similar protein profiles carried out by SDS-PAGE. Calcium transport in untreated vesicles and those treated with different concentrations of saponin seemed to have the same quantitative and qualitative parameters if the saponin was used in a range between 10 and 50 micrograms ml-1. Our results confirm that saponin may be considered to be a valid skinning agent for the external membranes of fast skeletal muscles.

Cyclic nucleotides and haemoglobin concentration in rabbit bone marrow cell suspensions stimulated by purified erythropoietin.

This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep). The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions. The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db-cAMP) or dibutyryl cGMP (db-cGMP), respectively. The following results were obtained: (1) upon the addition of 1 IU ml-1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml-1 Ep was present in the incubation mixture. (2) A mimetic effect on Hb concentration was obtained upon the addition of db-cAMP or db-cGMP to the suspensions. (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions. This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.