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Owing to close associations of different chromosomes at their heterochromatic regions, nuclear repatternings consisting of changes in the number and size of the chromocenters may occur with tissue differentiation in plant species. By studying these changes in the nuclear structure in a number of Magnoliopsida with chromocentric nuclei, we found: (i) that chromocenter association is affected by the degree of partitioning of the genome, since the extent of the phenomenon correlates positively with the number of chromosomes in the complement, the level of ploidy, and the number of chromocenters in meristematic nuclei, whereas the correlation with the mean DNA content per chromosome is negative; (ii) that this kind of nuclear repatterning plays a role in or is a marker of events that have a part in the control of nuclear activity, since a negative correlation was found to exist, as a rule, between the extent of chromocenter association and that of RNA synthesis in the nucleus; and (iii) that this large-scale control mechanism of gene expression is exploited in cell differentiation from its early stages. Indeed, chromocenter association takes place at the base of the meristems and remains unchanged during further tissue development. In certain species, this phenomenon has not been observed; its presence or absence may represent a character of entire families, and may be linked to the position of a species on an evolutionary scale.

Heat-Shock Protein 70 (HSP70) is a class of highly conserved proteins which is involved in essential functions as molecular chaperones and in the acquired tolerance processes. In this work, two cDNAs encoding a constitutive Hsc70 and an inducible Hsp70 from the water frog Rana (Pelophylax) lessonae, belonging to the Rana (P.) esculenta complex of central Italy, have been isolated and characterized. The two cDNA clones, named Rl-Hsc70 and Rl-Hsp70, encode 646 and 640 amino acid proteins respectively, which present extremely conserved functional domains characteristic of cytosolic members of the HSP70 family. Comparative studies of the amino acid sequences showed that Rl-Hsc70 and Rl-Hsp70 had the highest homology with constitutive and inducible HSP70 members of other amphibian species. The phylogenetic analysis also demonstrated a separate clustering of the Rl-Hsc70 and Rl-Hsp70 with constitutive and inducible members from other vertebrate species. Heat-inducibility assays performed during embryogenesis showed that the two isolated mRNAs displayed different expression profiles. Rl-Hsp70 was induced only in heat shock-treated embryos, whereas Rl-Hsc70 transcript levels, which were constitutively modulated in non-stressed embryos, did not increase following the heat treatment. In situ hybridization experiments demonstrated that both transcripts showed a tissue-specific enrichment in the central nervous system and in the somites.

Concentrations of commonly used organochlorine pesticides (OCPs) were determined in tissues of 23 adult and 24 larval water frogs of two coexisting species (Rana lessonae and the hemiclonal hybrid R. esculenta) and in the water of their breeding pond in an agricultural zone in Umbria, central Italy, where increased occurrence of infectious diseases and distinctly oversized tadpoles were recently observed. The concentrations of OCP in tissues of both species were lower than those in the water of their breeding pond, except for DDT, which was more concentrated in adult frogs than in pond water (bioaccumulation factor 7 for R. lessonae, 15 for R. esculenta). Total OCP concentration and adult body weight were positively correlated for both species, which is consistent with bioaccumulation. In accord, adults contained higher OCP concentrations than tadpoles. Oversized tadpoles had higher OCP concentrations than normal tadpoles. Mean OCP concentrations in individual organs were about an order of magnitude higher than those in whole-frog homogenates. They were highest in brain, higher in ventral than in dorsal skin, and moderately high in ovaries; transmission of bioaccumulation loads to the next generation is therefore possible. The observed OCP concentrations appear too low to directly cause mortality in water frogs, but effects of cumulative exposure to low-level pollutants and their synergistic interactions with the effects of other natural and anthropogenic environmental stressors are unknown.

Some chemical compounds used in intensive agriculture have been found to induce estrogenic effects; therefore a histological analysis of the testes and an evaluation of plasma levels of sex steroid, thyroid hormones, and vitellogenin were carried out in adult male water frogs of two coexisting taxa (Rana lessonae and the hemiclonal hybrid Rana esculenta) sampled in agricultural and pristine areas. Differences in seasonal profiles of hormones were found in water frogs living in the agricultural area where the presence of endocrine disrupting compounds was suspected on the basis of a previous study. In R. esculenta, sampled in the pristine area, high androgen levels were found in May; the opposite trend was found for R. esculenta sampled in agricultural areas in which the highest androgen levels were found in September, significantly lower compared with those found in R. esculenta sampled in the pristine area. Low androgen levels were also recorded in R. lessonae males sampled both in pristine and agricultural areas, while the highest levels were found in September. Regarding the trend of estradiol-17beta, an increase of this hormone was found in July both in esculenta and lessonae sampled in the agricultural area, and in the same month an estradiol-17beta peak, even though lower, was also found both in esculenta and lessonae males captured in the pristine area; detectable vitellogenin was found neither in males captured in the agricultural area, nor in those sampled in the pristine one. Moreover, while no significant changes of thyroid hormones were found either in the esculenta or lessonae males sampled in the pristine area, increased T3 and T4 titers were found in July in both esculenta and lessonae captured in the agricultural area. Morphological differences of the testes in males of parental species captured in the agricultural area were also observed. These findings indicate alterations in endocrine and reproductive function in frogs in the agricultural area, that could suggest the presence of endocrine disrupting compounds.