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Heavy metal pollution has become one of the most serious environmental problems. They can make DNA damage, due to their ability to form reactive oxygen species, causing mutations in human cells which may cause tumors. In order to monitor sea levels of heavy metals are used specific bioindicators like Mytilus galloprovincialis whose sperm chromatin is organized by three protamine-like (PLII, PLIII and PLIV), which belong to sperm nuclear basic proteins. We report the effects of Cd2+ on DNA, PLII and PLIII. Both PLII and PLIII acquired a slight proteinase K digestion resistance in the presence of metal. We also analyzed the self-association ability of PLII and PLIII Cd2+mediated. Turbidity assay showed that cadmium caused PLII but not PLIII aggregation, while glutaraldehyde produced a reduction of self-association ability for both proteins. Further, Cd2+ induced a decreasing of PLII DNA binding affinity but an increasing for PLIII and promoted H2O2 DNA damage even in the presence of Protamine-like. Those effects may result in an incorrect chromatin packaging that could decrease Mytilus galloprovincialis’ fitness and that of other marine species exposed to this polluting agent.

We have analyzed Mytilus galloprovincialis’ sperm chromatin which consists of three sperm nuclear basic proteins of the PL-type: PL-II, PL-III and PL-IV, in addition to a residual amount of the four core histones. The knowledge of Mytilus galloprovincialis’ sperm chromatin structure, the organization of the PL-type component in chromatin and their interactions with DNA is of considerable interest because these organisms are largely utilized as bioindicator of marine pollution that could affect its state. Our results obtained by micrococcal nuclease digestion in combination with salt fractionation suggest the existence of a likely unusual organization in which there would be a more accessible location of PL-II/PL-IV when compared to PL-III and core histones. Further we used electrophoretic mobility shift assay in order to define DNA binding mode of PLII and PLIII and turbidimetric assays to determine their self-association ability in the presence of sodium phosphate. On the base of our results we propose a model of Mytilus galloprovincialis’ sperm chromatin organization that could be useful in order to develop chromatin-based genotoxicity tests in pollution biomonitoring programs.

In sperm cells, DNA is complexed with sperm nuclear basic proteins (SNBPs) to form chromatin. Although SNBPs protect DNA from a variety of potentially dangerous reactive species, such as hydroxyl radicals (•OH), chromatin packaging doesn’t protect DNA from metal ion-dependent damage in the presence of hydroxyl radicals. We report the effects induced by Cu2+ and Ni2+ ions on DNA and Mytilus galloprovincialis’ protamine-like II and III. Protamine-like III acquires proteinase k digestion resistance in the presence of copper and not in the presence of nickel; PLII, in contrast with PLIII, is sensitive to proteinase k digestion in presence of both metals. Further, copper promotes hydrogen peroxide damage of DNA also in the presence of PL-types while nickel promoting PL-types aggregation enhances DNA compactness and protection. Nickel in fact, in contrast to copper, increases PLII and PLIII DNA binding affinity. All these effects can lead an anomalous chromatin packaging and, therefore, a reduction of Mytilus galloprovincialis’ reproductive fitness and of other organisms exposed to the same toxicants.

Heavy metal pollution has become one of the most serious environmental problems in the world that brought threat to human health and ecological security. To monitor sea levels of heavy metals we used as specific bioindicator Mytilus galloprovincialis whose sperm chromatin is organized mainly by three protamine-like proteins (PLII, PLIII and PLIV). These proteins protect DNA from a variety of potentially dangerous reactive species but chromatin packaging doesn’t protect DNA from metal ion-dependent damage in the presence of hydroxyl radicals. We report the effects induced by Cu2+, Ni2+ and Cd2 ions on DNA and on Mytilus galloprovincialis’ PLII and PLIII. Self-association ability of PLII and PLIII mediated by Cd2+, Ni2+ and Cu2+ show that Cd2+ causes only PLII aggregation to a greater extend respect to Ni2+ that induces a mild aggregation of both PLII and PLIII; Cu2+, instead, doesn’t produce any type of aggregates. In vivo studies show that these heavy metals affect PLII, PLIII and PLIV state and properties. While nickel produces an increase of DNA binding affinity for both PLII and PLIII cadmium causes a decrease of PLII DNA binding affinity but an increase for PLIII. Copper and cadmium promote hydrogen peroxide damage of DNA also in the presence of protamine-like proteins while nickel doesn’t. All these effects can lead an anomalous chromatin packaging and a reduction of the reproductive fitness in Mytilus galloprovincialis and other organisms exposed to the same toxicants.

Supercoiled state corresponds to the active form for plasmid applications. The relaxed circular form of plasmids is often inactive or poorly active. To obtain significant amounts of almost fully supercoiled DNA, we modified the standard protocol of a commercially available Qiagen plasmid purification kit. Our changes led to isolation of almost 100% of the plasmids in the supercoiled state. The modified protocol was used to purify different plasmids with consistent results. The purified plasmids maintain supercoiled state for about two months. The modified protocol is very advantageous because it allows easy DNA production with high degree of supercoiled form at low cost.

Obesity is associated with an increased risk of breast cancer. A number of adipocytokines are increased in obesity causing low-level chronic inflammation associated with an increased risk of tumors. The adipocytokine leptin shows profound anti-obesity and pro-inflammatory activities. We have hypothesized that in common obesity, high circulating leptin levels might contribute to an increased risk of breast cancer by affecting mammary cell proliferation and survival. Leptin exerts its activity not only through leptin receptor (LepR), but also through crosstalk with other signaling systems implicated in tumorigenesis. In this study, we focused our attention on the relationship between the leptin/LepR axis and the estrogen receptor-alpha (ERalpha). To this aim, we utilized two human breast cancer cell lines, one ERalpha-positive cell line (MCF 7) and the other ERalpha-negative cell line (MDA-MB 231). We observed that the two cell lines had a different sensitivity to recombinant leptin (rleptin): on MCF 7 cells, rleptin induced a strong phosphorylation of the signal transducer and activator of transcription (STAT) 3 and of the extracellular related kinase 1/2 pathways with an increased cell viability and proliferation associated with an increased expression of ERalpha receptor. This response was not present in the MDA-MB 231 cells. The effects induced by leptin were lost when LepR was neutralized using either a monoclonal inhibitory antibody to LepR or LepR gene-silencing siRNA. These data suggest that there is a bidirectional communication between LepR and ERalpha, and that neutralization and/or inactivation of LepR inhibits proliferation and viability of human breast cancer cell lines. This evidence was confirmed by ex vivo studies, in which we analyzed 33 patients with breast cancer at different stages of disease, and observed that there was a statistically significant correlation between the expression of LepR and ERalpha. In conclusion, this study suggests a crosstalk between LepR and ERalpha, and could envisage novel therapeutic settings aimed at targeting the LepR in breast cancers.

The mode of binding of sperm and somatic H1 histones to DNA has been investigated by analyzing the effect of their addition on the electrophoretic mobility of linear and circular plasmid molecules. Low concentrations of sperm histones do not appear to alter the electrophoretic mobility of DNA, whereas at increasing concentrations, an additional DNA band is observed near the migration origin. This band then becomes the only component at higher values. In contrast, somatic histones cause a gradual retardation in the mobility of the DNA band at low concentrations and aggregated structures are observed only at higher values. Experiments on the H1 globular domain obtained by limited proteolysis indicate that the mode of binding to DNA depends on the H1 globular domain. The arginine residues appear to be relevant for the different effects as indicated by experiments on sperm histone and on protamine with arginines deguanidinated to ornithines. The modified molecules influence DNA mobility like somatic H1s, indicating that the positive guanidino groups of arginines cannot be substituted by the positive amino groups of ornithines. Modifications of the amino groups of lysines show that these residues are necessary for the binding of H1 histones to DNA but they have no influence on the binding mode.

Self-association of histones H1 from calf thymus and from sperm of the marine worm Chaetopterus variopedatus was studied on native and glutaraldehyde cross-linked molecules by PAGE and by salt-induced turbidity measurements. Multiple polymers were generated by native sperm histone H1-like after glutaraldehyde cross-linking while the same treatment on its lysine- or arginine-modified derivatives and on somatic histone H1 failed to induce polymerization. This result suggests the relevance of arginine content in the formation of histone H1-like polymers particularly because Chaetopterus variopedatus and calf thymus histones H1 have similar content of lysine but different K/R ratio (2 and 15, respectively). Salt-induced turbidity experiments confirmed the high tendency of sperm histone H1-like to form oligomers, particularly in the presence of phosphate ions. Native PAGE analysis in the presence of phosphate supported this hypothesis. The reported results suggest that phosphate ions connecting lysine and arginine side chain groups contribute to the interaction of sperm histone H1-like with DNA in chromatin and play a key role in organization and stabilization of the chromatin higher order structures.

A novel gene encoding a new H3 histone variant (H3L) has been identified in P. lividus sea urchin embryo. It encodes a H3 histone protein showing the S.//.A.IG amino acid motif typical of the replication independent H3.3 variants but in a mRNA showing the 3′ terminal stem-loop nucleotide sequence that is typical of the replication dependent variants. The gene is intronless, the corresponding short transcript is non-polyadenylated and its expression is replication dependent with a timing of a late variant. The new H3 variant is expressed as a minor component with respect to a major replication dependent late H3 histone here identified by partial cDNA sequence. These results show that classification of histones in replication dependent and independent variants only on the basis of their amino acid sequences should be reconsidered.