n-TiO2 and CdCl2 co-exposure to titanium dioxide nanoparticles and cadmium: Genomic, DNA and chromosomal damage evaluation in the marine fish European sea bass (Dicentrarchus labrax).

Due to the large production and growing use of titanium dioxide nanoparticles (n-TiO2), their release in the marine environment and their potential interaction with existing toxic contaminants represent a growing concern for biota. Different end-points of genotoxicity were investigated in the European sea bass Dicentrarchus labrax exposed to n-TiO2 (1mgL(-1)) either alone and combined with CdCl2 (0.1mgL(-1)) for 7 days. DNA primary damage (comet assay), apoptotic cells (diffusion assay), occurrence of micronuclei and nuclear abnormalities (cytome assay) were assessed in peripheral erythrocytes and genomic stability (random amplified polymorphism DNA-PCR, RAPD assay) in muscle tissue. Results showed that genome template stability was reduced after CdCl2 and n-TiO2 exposure. Exposure to n-TiO2 alone was responsible for chromosomal alteration but ineffective in terms of DNA damage; while the opposite was observed in CdCl2 exposed specimens. Co-exposure apparently prevents the chromosomal damage and leads to a partial recovery of the genome template stability.

Genomic and chromosomal damage in the marine mussel Mytilus galloprovincialis: Effects of the combined exposure to titanium dioxide nanoparticles and cadmium chloride.

Titanium dioxide nanoparticles (TiO2-NPs) continuously released into waters, may cause harmful effects to marine organisms and their potential interaction with conventional toxic contaminants represents a growing concern for biota. We investigated the genotoxic potential of nanosized titanium dioxide (n-TiO2) (100 µg L(-1)) alone and in combination with CdCl2 (100 µg L(-1)) in Mytilus galloprovincialis after 4 days of in vivo exposure. RAPD-PCR technique and Micronucleus test were used to study genotoxicity. The results showed genome template stability (GTS) being markedly reduced after single exposure to n-TiO2 and CdCl2. Otherwise, co-exposure resulted in a milder reduction of GTS. Exposure to n-TiO2 was responsible for a significant increase of micronucleated cell frequency in gill tissue, while no chromosomal damage was observed after CdCl2 exposure as well as after combined exposure to both substances.

RAPD-PCR analysis for molecular characterization and genotoxic studies of a new marine fish cell line derived from Dicentrarchus labrax.

Continuous cell lines could provide an important tool for studying epidemiology, toxicology, cellular physiology and the host-pathogen interactions. Random amplified polymorphic deoxyribonucleic acid analysis by PCR (RAPD-PCR) was used for the molecular characterization of Dicentrarchus labrax embryonic cells (DLEC) as a possible tool to detect DNA alterations in environmental genotoxic studies. We studied the DNA pattern of the DLEC fish cell line, a fibroblast-like cell line derived from European sea bass. From a total of 15 primers only six showed good discriminatory power for the amplification process on DNA samples collected from cells by three different methods (organic extraction, salting-out method and chelating agent extraction). The results obtained show that the cell line chosen for this study could be used as a possible tool for the detection of potential genotoxicity of numerous chemical compounds.

Molecular and chromosomal analysis of ribosomal cistrons in two cartilaginous fish, Taeniura lymma and Raja montagui (Chondrichthyes, Batoidea).

We used silver nitrate staining, CMA3 and FISH to study the chromosomal localization of both the major ribosomal genes and the nucleolar organizer regions as well as that of the minor ribosomal genes (5S rDNA) in two species of Batoidea, Taeniura lymma (Dasyatidae) and Raja montagui (Rajidae). In both species, all the metaphases examined showed the presence of multiple NOR-bearing sites, while the gene for 5S rRNA proved to be localized on two chromosome pairs. Furthermore, one of the two 5S rDNA sites in T. lymma was shown to be co-localized with the major ribosomal cluster. The presence of multiple nucleolar organizer regions in the two species might be interpreted as being the result of intraspecific polymorphisms, or as a phenomenon of the amplified transposition of mobile elements of the genome. We also determined the nucleotide sequence of the 5S rRNA gene, consisting of 564 bp in R. montagui and 612 bp in T. lymma. We also found TATA-like and (TGC)n trinucleotides, (CA)n dinucleotides and (GTGA)n tetranucleotides, which probably influence gene regulation.

Chromosomal mapping and molecular characterization of ribosomal RNA genes in Lebias fasciata (Teleostei, Cyprinodontidae).

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.

Selachian cytogenetics: a review.

The karyotype of Chondrichthyes is still the least investigated among vertebrates. Over the last 40 years, the karyotypes of 63 out of the 1100 known species (5.73%) have been described in literature, namely seven squalomorph, one squatinomorph, 20 galeomorph, 33 batoid and two holocephalian species. Generally, the diploid number ranges from a minimum of 28 to a maximum of 106 elements, with more frequent values observed between 50 and 100 chromosomes. None of the four superorders is characterized by a peculiar chromosome set or morphology; the number of uniarmed and biarmed elements is variable in all the karyotypes, and microchromosomes are often present. The general trend in all groups seems to be a progressive reduction of the telocentric chromosome number in the most specialized species, followed by the loss of the microchromosomes. Polyploidy, followed by diploidization events and Robertsonian rearrangements, might have played a key role in the karyological evolution of elasmobranch fish. Chondrichthyes have the largest genome sizes among vertebrates, with the exception of dipnoans and urodeles. In the whole class, the species examined vary greatly in size, from 3 to 34pg/N: the lowest values have been observed in holocephalians, while galeoids and batoids have a DNA amount ranging from 5 to 15 pg/N. Squaloids show heterogeneous DNA amounts, ranging from 8 to 34 pg/N. In more recent years, karyological studies have provided new data on the characterization of selachian karyotypes by C-banding, NOR staining, restriction enzymes in situ digestion and FISH with specific DNA probes, such as telomeric and SINE sequences.

(TTAGGG)n telomeric sequence in selachian chromosomes.

The distribution of telomeric sequence (TTAGGG)n in the genomes of Chondrichthian species at different stages of evolution was investigated both by DNA genomic hybridization, and by fluorescence in-situ hybridization (FISH) of metaphase chromosomes. The sequence is highly conserved in all the species examined. FISH revealed a label uptake only by the telomeres in Raja asterias. However, in Torpedo ocellata, we revealed pericentromeric and interstitial sequence localization on some chromosome pairs in addition to the hybridization signal on telomeres. These findings confirm that the karyotype evolution of these fish began by Robertsonian fusion.

The history of science: defending epistemology with new technologies.

The leap in scientific discoveries made with new computer technologies can be compared to the invention of the printing press and expanding scientific thought in the 16th century. Nowadays, new communication technologies offering a multimedia contribution illustrate the fascinating reality of scientific exploration even for individuals who can otherwise not be reached. Since rare books are usually kept in libraries with limited access, the strict safekeeping, however, often overrides the need for academic consultation. Moreover, rare manuscripts are often illustrated with figures that are just as important as the written texts, but which are also unavailable. We combined our expertise in biology, medicine and multimedia to produce a CD-ROM with a hyperlinked text on the scientific revolution of the 16th century, focusing on the early conceptualization of life sciences in Europe. In this paper we show the first page of Malpighi's Epistle II (De pulmonibus - on the Lungs) as it appeared in Opera Omnia published in London (1686) by the Royal Society.

Copresence of Deleted Protein Species Generates Structural Heterogeneity of Ovine alpha(s1)-Casein.

Multiple forms of mature alpha(s1)-casein have been characterized in ovine variants A and D using a combination of mass spectrometry and automated Edman degradation. Mature ovine alpha(s1)-casein was found to be a heterogeneous mixture of at least seven molecular species. The main component, representing about 50% total alpha(s1)-casein, corresponded to the full-length (199 residues long) protein. The other components were alpha(s1)-casein of different lengths: 198 (less Gln78), 191 (less peptide 110-117), 191 residues (less peptide 140-148), 190 (less peptide 110-117 and Gln78), 190 (less peptide 140-148 and Gln78), and 183 (less peptides 110-117 and 140-148) residues long alpha(s1)-casein. Each of the alpha(s1)-casein multiple forms occurred at three different phosphorylation levels, due to the partial phosphorylation of both Ser115 (at about 50%) and Ser41 (at about 20%). In the case of deleted peptide 110-117, the protein heterogeneity linked to the partially phosphorylated Ser115 was abolished, and only two levels of phosphorylation were observed. These multiple forms differing in molecular weight and degree of phosphorylation may have been developed from an exon skipping during mRNA splicing in ovine alpha(s1)-casein, similar to that recently described in the case of its caprine counterpart.

Cloning and characterization of a repetitive DNA detected by HindIII in the genome of Raja montagui (Batoidea, Chondrichthyes).

A repetitive HindIII fragment of DNA from Raja montagui (Rajiformes) was cloned and sequenced for the first time in cartilaginous fishes. This element, which comprises approximately 5% of the whole genome of the spotted ray, is absent in long tandem arrays, being typical of satellite DNA. It appeared constituted by 311 AT-rich bp (61%). The clone was hybridized to the genomic DNA of species with varying phyletic distances, revealing a high degree of conservation.

NOR and heterochromatin analysis in two cartilaginous fishes by C-, Ag- and RE (restriction endonuclease)-banding.

Selachian chromosomes were investigated for the first time by C-, Ag- and RE (restriction endonuclease)-banding. A pair of NOR-bearing acrocentric chromosomes, as well as a third extra site, were demonstrated in two species of Torpedo. In situ digestion with restriction endonucleases revealed a peculiar arrangement of constitutive heterochromatin: in Torpedo ocellata the centromere never appeared C-banded, and was always digested by AluI, PstI, DdeI and HaeIII; in Torpedo marmorata, instead, heterochromatin was present in the centromere, and was not digested by the same enzymes. The occurrence of different types of centromeric heterochromatin suggests an involvement of highly repeated DNA fractions in the Robertsonian phenomena that occurred in chromosome rearrangements in the genus Torpedo.

Chromosome markers and karyology of selachians.

Among vertebrates, chondrichthyans exhibit peculiar karyotypes and total amount and composition of DNA very different from those of tetrapods and teleosteans. Selachians have relatively large genome sizes (more than 30 pg/N), which are inversely related to the fraction rich in adenine-thymine. Moreover, they show a high chromosome number (2n = 60-100), decreasing in the most specialized species. The karyotypes of Scyliorhinus stellaris, Torpedo ocellata, and T. marmorata have been investigated by several techniques in order to distinguish particular genome fractions along the chromosome arms. C-banding appears to be positive in most telomeric regions in Scyliorhinus and at the interstitial level in the two torpedo species. Studies with restriction enzymes (RE) have just been started in selachians, employing ALU I and HIND III in Scyliorhinus stellaris. The former digests the whole chromosome except the telomeric regions, revealing patterns similar to the C-bands. The latter cuts the chromosomes into several interstitial regions, producing G-bands. Other RE are being studied, which will allow identification in situ of qualitative differences in the various DNAs.

Sequence organization in the DNA of three Selachians.

The DNA interspersion pattern in 3 Selachians (R. asterias, T. marmorata and S. stellaris) has been studied through the reassociation kinetics of short (0.3 Kb) and long (2.5 Kb) DNA fragments. Preliminary results show that most of the DNA (approximately 80%) of these organisms is arranged according to a short-period interspersion pattern. A notable resemblance to the pattern previously described in the teleostean Salmo trutta has been observed.

Repetitive DNA and polyploidy in selachians.

1. The DNA reassociation kinetics have been studied in 6 selachian species: Raja asterias, Raja montagui, Dasyatis violacea, Torpedo marmorata, Torpedo ocellata and Oxynotus centrina. 2. The results obtained show that the genomes of the two Torpedo, Dasyatis and Oxynotus are polyploid if compared with those of the two rays, though this finds no correspondence in the diploid chromosome number. 3. The phenomena of polyploidization would often be followed by wide chromosome rearrangements and by a progressive divergence of the various repetitive DNA sequences. 4. The existence of polyploidy in almost all the main superorders of living Selachians suggests that this mechanism of genomic evolution may have played an important role in the phylogeny of this class.