Gian Luigi Gianfranceschi

Comparative activity of antioxidants from wheat sprouts, Morinda citrifolia, fermented papaya and white tea

Hydroalcoholic extracts from wheat sprouts, white tea, Morinda citrifolia and fermented papaya were analysed to determine their reducing power and antioxidant activity. The results show that the micromoles of potassium ferricyanide reduced by a quantity of extract corresponding to 1 g of the various dehydrated starting tissues are: 12.91±0.83 (wheat sprouts), 10.66±1.22 (M. citrifolia), 17.06±1.24 (white tea), and 1.05±0.09 (fermented papaya). In addition the results show a strong oxygen superoxide scavenging activity in the extracts from white tea, M. citrifolia and wheat sprouts. The activity of the fermented papaya extract is the lowest. The thin-layer chromatography and UV spectrophotometry of the extracts show in each source a mixture of antioxidant compounds probably belonging to the families of reducing glycosides and polyphenols. The chromatographic pattern of the antioxidant compounds and the UV spectrum are quite different in the various sources.

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.

Nutritional relevance of wheat sprouts containing high levels of organic phosphates and antioxidant compounds

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of antioxidant molecules. Catalase and peroxidase activity appears very strong. Regarding low-molecular weight antioxidant molecules, TLC and other biochemical analysis show the presence of several classes of antioxidant compounds such as reducing glycosides, polyphenols, and –SH groups. Antioxidant activity has been measured by potassium ferricyanide reduction and by means of superoxide scavenging NBT. It has also been observed that the oral assumption of wheat sprout powder induces in old dogs a significant reduction of senile cataract. These results clearly confirm that the administration of natural antioxidant compounds can prevent or delay senile cataract. Moreover, it is evident that wheat sprout biologically active substances can be at least partially absorbed during the digestion process. Our research is now directed to the recovery of large amounts of wheat sprout biologically active substances that could also be used in the formulation of nutritional complements.

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.

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.

Acidic pentapeptide phosphorylated in vitro by calf thymus protein kinase NII binds to DNA in the presence of Mg2+ cations

The pentapeptide pyroGlu‐Ala‐Glu‐Ser‐Asn has been synthetized and phosphorylated in vitro at level of serine by protein kinase NII isolated from calf thymus chromatin. It is noteworthy that the calf thymus kinase NII shows a remarkable affinity for this peptide. The [32P]peptide is able to bind to several DNAs in the presence of Mg2+ (λ phage, calf thymus, pBR540 plasmid). This binding appears not specific with regard to the type of DNA and its base sequence. These data support the hypothesis that phosphorylated acidic domains of nuclear nonhistone proteins could bind directly to DNA in the presence of Mg2+ cations

Phosphorylation sites for type n ii protein kinase in DNA-topoisomerase i from calf thymus

1. Calf thymus DNA-topoisomerase I has been isolated, in an improved preparation, nearly to SDS-PAGE homogeneity, as a single major protein (100 kDa). 2. In vitro labeling experiments, which employed the purified enzyme [gamma-32P]ATP and N II protein kinase, also showed that the calf thymus topoisomerase I became phosphorylated. 3. Phosphorylation was accompanied by an increase in topoisomerase I activity. 4. Phosphoaminoacid analysis indicated that only serine residues became phosphorylated. 5. Tryptic peptides mapping, by HV electrophoresis, identified five major [32P]peptides. This number is higher than that reported for topoisomerase I from Novikoff hepatoma cells. 6. Separation of each spot, by reverse phase HPLC, resulted in their elution at fractions 1, 2, 3, 4 and 5 with 9, 11, 16, 27 and 28% acetonitrile, respectively. 7. Isolated phosphopeptides will be subjected to sequencing, to DNA-binding and transcription regulation tests; then, it will be speculated whether type N II protein kinase may contribute to the physiological regulation of DNA topoisomerase I activity from calf thymus, as well.

Short acidic peptides isolated from wheat sprout chromatin and involved in the control of cell proliferation. Characterization by infrared spectroscopy and mass spectrometry.

Low molecular weight peptides were isolated from the chromatin of wheat sprouts. Following gel filtration the peptide fraction shows a sharp inhibiting activity on the growth of HeLa cancer cells. Infrared (IR) spectroscopy and mass spectrometry have been utilized to characterize the wheat sprout peptides in an attempt to recognize the peptide sequence involved in the control of cell growth. The quantitative presence of a peptide with MH+=572 appears proportional to the cell growth inhibition activity. This compound has been subjected to extensive mass spectrometry analysis. The automatic computational analysis of the ions of second, third and fourth generations indicate a peptide sequence, AcHis-Asp-Ser-Glu-, that binds at the C-terminal a molecule of ethanolamine. Moreover, the results show that some sequences of the wheat sprout peptide family are present in the peptide fractions isolated from several other tissues, thus supporting the hypothesis of ubiquitous regulatory peptides.

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.

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.