Marina Piscopo

Defensome against Toxic Diatom Aldehydes in the Sea Urchin Paracentrotus lividus

Many diatom species produce polyunsaturated aldehydes, such as decadienal, which compromise embryonic and larval development in benthic organisms. Here newly fertilized Paracentrotus lividus sea urchins were exposed to low concentration of decadienal and the expression levels of sixteen genes, implicated in a broad range of functional responses, were followed by Real Time qPCR in order to identify potential decadienal targets. We show that at low decadienal concentrations the sea urchin Paracentrotus lividus places in motion different classes of genes to defend itself against this toxic aldehyde, activating hsp60 and two proteases, hat and BP10, at the blastula stage and hsp56 and several other genes (14-3-3ε, p38 MAPK, MTase, and GS) at the prism stage. At this latter stage all genes involved in skeletogenesis (Nec, uni, SM50 and SM30) were also down-expressed, following developmental abnormalities that mainly affected skeleton morphogenesis. Moreover, sea urchin embryos treated with increasing concentrations of decadienal revealed a dose-dependent response of activated target genes. Finally, we suggest that this orchestrated defense system against decadienal represents part of the chemical defensome of P. lividus affording protection from environmental toxicants.

Antimicrobial activity of various cationic molecules on foodborne pathogens

Antibacterial effects of various arginine- and lysine-rich polycationic proteins and polymers were evaluated by broth and solid dilution assay on a range of foodborne pathogens, Gram-positive and Gram-negative bacteria. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of α-poly-l-lysine (poly-lys), α-poly-l-arginine (poly-arg) and protamines from herring sperm (clupeine sulphate) and salmon sperm (salmine sulphate) were determined on Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella sonnei, Escherichia coli O157:H7 and Pseudomonas aeruginosa. All these molecules showed antibacterial activity on all strains with different MIC and MBC values. The molecular mechanisms underlying the effect of α-poly-l-arginine might be related to the entrance of the molecule into the cell. In fact α-poly-l-arginine labelled with 7-Diethylamino coumarin-3-carboxylic acid, succinimidyl ester (DEAC,SE) showed ability to permeate the cell membrane of B. cereus and E. coli O157:H7.

Epigenetic chromatin modifications in the cortical spreading depression.

Preconditioning with Cortical Spreading Depression induces a sort of tolerance to a subsequent episode of ischemia. The mechanism of this tolerance is not clear. We studied if such treatment induces epigenetic chromatin modifications on the hemispheres of rats preconditioned by Cortical Spreading Depression. The contralateral hemispheres were used as control. We determined the level of H3K4 and H3K9 methylation and the mRNA amounts for the two well known H3K4 methyltransferases (MLL and SET7) in rats 24 degrees h after the Cortical Spreading Depression induction. Western blotting experiments have been performed using three different types of primary antibodies against mono-, di- and tri-methyl H3K4 and primary antibody anti-dimethyl H3K9. In the same samples we checked if the H3 histones were replaced by the H3.3 histone variants that could be an additional marker of chromatin modifications. The level of mono- and di-methyl H3K4 was significantly lower in samples of the treated hemispheres than those of the contralateral hemispheres (40% and about 60%, respectively) while the level of tri-methylation remained unchanged. The level of di-methyl H3K9 was almost 60% higher in the treated hemispheres than the contralateral hemispheres. The treatment for Cortical Spreading Depression affected also the level of expression of H3K4 histone methyltransferase MLL and SET7 that decreased in the treated hemispheres in comparison to the control hemispheres (80% and 40%, respectively). The treatment for Cortical Spreading Depression instead had no effects on the overall amounts of mRNA for H3 and H3.3 histones. In conclusion epigenetic chromatin modifications are evident in rats 24 degrees h after the Cortical Spreading Depression induction.

Localization of calbindin D-28K in the otoconia of lizard Podarcis sicula.

The membranous labyrinth of lizard Podarcis sicula contains calcite and aragonite crystals. Saccule, utricle and lagena contain calcite crystals while aragonite crystals are present only in the saccule where they are very abundant. We have recently demonstrated the presence of calbindin D-28K in the organic matrix of lizard P. sicula otoconia. In order to define its localization, since calbindin modulates cellular Ca2+ level, otoconia from utricle and lagena were collected separately from those from saccule and then otoconial proteins were extracted. Immunoblot assay on proteins extracted from the otoconia and confocal laser scanning microscope analyses of otoconia using monoclonal anti-calbindin D-28K antibodies indicated that calbindin D-28K is a protein typical of aragonite crystals.

Calbindin D28K is a component of the organic matrix of lizard Podarcis sicula otoconia

The factors controlling otoconia growth are not well known but it seems that the type of proteins contained in the otoconia regulates the initiation and/or the subsequent rates of crystal growth determining the morphology and the size of the final crystal. In order to clarify the mechanism of otoconia formation and their turnover, major proteins contained in the otoconia from the maculae of the saccule, utricle and lagena of inner ear of lizard Podarcis sicula were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie staining of SDS-PAGE resulted in a major broad band of 15 kDa and four other bands of 21, 28, 45 and 97 kDa. The proteins of 15, 21, 28 and 45 kDa were separated by high-pressure liquid chromatography on a C-4-reverse-phase column and the incubation of blots with monoclonal anti-Calbindin D28K antibodies indicated that the band of 28 kDa was Calbindin D28K, a calcium-binding protein.

Anion‐mediated lysine—arginine interaction evidence in Chaetopterus variopedatus sperm protamine

Chaetopterus ariopedatus sperm protamine is a stable oligomer. Specific amino acid side chain modifications show that the oligomeric structure depends on anion‐mediated lysine—arginine interactions. The occurrence of this type of interaction is confirmed by the finding that Poly‐l‐arginine readily forms aggregates with Poly‐l‐lysine or with the native but not with the protamine with carbamylated ε‐amino groups.

Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis Kinetic and spectroscopic evidence

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.O. Protonation of the proximal imidazole at low pH (≤ 3.0) and the consequent cleavage of the HisF8NE2‐Fe proximal bond brings about a ≈ 20‐fold increase of the second‐order rate constant for CO binding. This process displays a pK a = 4.0 ± 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2‐Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH‐rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828–4830), can be described only by assuming a concerted proton‐linked transition with n = 1.8 ± 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.

Relevance of Arginines in the Mode of Binding of H1 Histones to DNA

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.

The biological response chain to pollution: a case study from the “Italian Triangle of Death” assessed with the liverwort Lunularia cruciata

The liverwort Lunularia cruciata, known for being a species tolerant to pollution able to colonize urban areas, was collected in the town of Acerra (South Italy) to investigate the biological effects of air pollution in one of the three vertices of the so-called Italian Triangle of Death. The ultrastructural damages observed by transmission electron microscopy in specimens collected in Acerra were compared with samples collected in the city center of Naples and in a small rural site far from sources of air pollution (Riccia, Molise, Southern Italy). The biological response chain to air pollution was investigated considering vitality, photosynthetic efficiency, heat shock protein 70 (Hsp70) induction and gene expression levels, and chlorophyll degradation and related ultrastructural alterations. Particularly, a significant increment in Hsp70 expression and occurrence, and modifications in the chloroplasts’ ultrastructure can be strictly related to the environmental pollution conditions in the three sites. The results could be interpreted in relation to the use of these parameters as biomarkers for environmental pollution.

A Sperm Nuclear Basic Protein from the Sperm of the Marine Worm Chaetopterus variopedatus with Sequence Similarity to the Arginine-Rich C-Termini of Chordate Protamine-Likes

The sperm nuclear basic proteins (SNBPs) of the marine annelid worm Chaetopterus variopedatus have been shown previously to consist of a mixture of two SNBPs: histone H1-like (CvH1) and C.variopedatus protamine-like (CvPL). Here, we report the structural characterization of CvPL. The protein has a molecular weight of 8370.5 Da, a K/R ratio of 0.34, and a secondary structure, which are intermediate between those of protamine (P) and protamine-like (PL) SNBPs. The N-terminal sequence of CvPL shows a high extent of similarity with the arginine-rich C-terminal domain of chordate PL-type SNBPs. Furthermore, the protein binds to DNA in a similar fashion as vertebrate PLs and their own CvH1, but in a way that is different from that of the lysine-rich somatic H1 histones. We have experimentally determined the molar ratio CvH1:CvPL to be ∼1:6 in C. variopedatus sperm. Based on all of these, a model is proposed for the organization of the sperm chromatin by CvH1 and CvPL.