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In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 μM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 μM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 μM CdCl2). We found also that, in response to a lethal dose of cadmium (1 μM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.

The functional role played by test cells in larvae of various ascidian species consists in depositing sub-microscopic structures known as ornaments and/or proteoglycan substances on the larval test surface. According to the data reported in the literature, the deposition of ornaments together with proteoglycan substances on the larval test would render the latter hydrophilic and thus allow the larva to swim being immersed in water. Ornament deposition on the larval test does not occur in all the ascidian species. Ultrastructural investigations made on larvae belonging to the Cionidae and Ascididae families, for instance, have failed to evidence the presence of ornaments on the test. For these ascidian families it has been hypothesized that in swimming larvae test cells secrete an amorphous substance that would allow them to adhere to the larval test. In order to ascertain the functional role played by test cells in swimming larvae of the Ascididae family, the presently reported ultrastructural and cytochemical investigations have been made on larvae of Ascidia malaca. Besides suggesting that test cells, tightly adherent to the test surface, present an amoeboidic behaviour, the ultrastructural investigations have evidenced that these cells are still metabolically active. Their cytoplasm, characterized by the presence of a Golgi apparatus actively involved in synthesis, is almost entirely filled with very large granules; some of them gradually empty their contents turning into vacuoles containing scarce residues of electrondense particles. The present ultrastructural observations support the hypothesis that the adhesion of test cells on the larval test could be very likely eased by the secretion of substances synthesized by the Golgi and released through pseudopodes which test cells then wedge into the test. The cytochemical investigations were based on a reaction (fixation in glutaraldehyde-tannic acid) which evidences the presence, at the ultrastructural level, of proteoglycan substances such as glycosaminoglycans (Singley and Solursh, 1980). The reaction has given positive results in test cell granules undergoing emptying, on the outer membrane of the same cells, and on the outer cuticular layer C1 of the larval test. The present investigations, besides confirming the absence of ornament deposition on the test surface by test cells of Ascidia malaca swimming larvae, have evidenced that the secretion products deposited on the larval test surface by test cells consist of glycosaminoglycans, i.e. proteoglycan substances. In agreement with the data reported in the literature, it is hypothesized that the deposition of glycosaminoglycans on the surface of Ascidia malaca larval test makes the larval tunic hydrophilic and thus the larva is able to swim being immersed in water.

The swimming larvae of ascidians are entirely covered by a hyalin coat called tunic, or test. This covering consists of two cuticular layers, C1 and C2, which surround an inner compartment composed of an amorphous hyalin matrix with numerous fibrils dispersed inside. Data from the literature agree on the key role played by the cells of the larval ectodermic layer in the synthesis and secretion of larval test components. In the present article are reported ultrastructural and cytochemical investigations made during test formation in the swimming larva of Ascidia malaca. Besides confirming the role played by ectodermic cells during the early stages of test formation, the investigations highlight the way in which the fibrillar component of the test is synthetized and secreted. At the ultrastructural level it has been evidenced that the C1 and C2 cuticular layers originate from the tight packing of fibrils. Based on the data reported in the present study, it is hypothesized that while a relevant part of the fibrils, once secreted, remains dispersed inside the matrix of the inner compartment of the test, quite likely in order to increase its consistency, packing of the remaining fibrils leads to the formation of the C1 and C2 cuticular layers. Packing of the fibrils in C1 and C2 could be favoured by their chemically adhesive nature. This hypothesis is strongly supported by the herewith reported results of the cytochemical investigations carried out on the test of the swimming larva of A. malaca. The cytochemical PA-TCH-SP reaction has in fact evidenced that both fibril types, i.e. those dispersed inside the inner compartment and those packed in the C1 and C2 cuticular layers, are constituted by glycoproteins and/or proteoglycans substances whose adhesive properties are well documented in the literature.

Background: Gallstone disease is a frequently encountered disorder in subjects living in Rosario. The reasons for the presence or absence of symptoms are unknown., Aims: To determine associations between biliary symptoms and ultrasonographic features of gallbladder and gallstones., Subjects: A random study was conducted on 1,173 subjects, inhabitants of 20 years and older, in the city of Rosario, Argentina., Methods: High-resolution abdominal ultrasound examinations were performed. Biliary pain was defined based on previously published definitions., Results: Gallstones were found in 149 subjects (101 female, 48 male) of whom 51% of females and 35% of males with cholelithiasis were symptomatic. Mean age was 53 years in symptomatic and 55 in asymptomatic subjects. Gallbladder size was normal in 97% of symptomatic and in 96% of the asymptomatic participants. There were no significant differences between the groups as far as concerns size and gallstone number. Impacted stones were observed in 10% of symptomatic and in none of the asymptomatic subjects (p<0.01)., Conclusions: Subjects' age and gender, gallstones size and number, as well as ultrasonographic features of gallbladder and biliary tract did not differ significantly between symptomatic and asymptomatic subjects. Only impacted stones were significantly more frequent in symptomatic subjects.

In the present work are reported investigations on the formation and chemical nature of the chorion of Ascidia malaca oocytes. The ultrastructural observations have shown that both follicle cells and test cells play a key role on chorion formation. At the beginning of vitellogenesis (stage I), chorion is formed by a single fibrogranular layer. During vitellogenesis (stage II), the chorion becomes, at first, bi-layered and successively presents three layers. The outer layer (ofl) is composed of a thin and irregular network of short fibrils. The central layer (cdl) is also made by a fibrillar component which is very compact to form an electron-dense layer. The inner layer (ifl) has a larger size compared to that of outer and central layer and it is composed by a matrix containing little and scattered globular electron-dense granules and several fibrils oriented in all directions. At the end of vitellogenesis beneath the three-layered chorion a large perivitelline space will be formed. In this space are found the test cells. Cytochemical investigations performed at light microscopy (toluidine blue) and electron microscopy (PA-TCH-SP) have shown in Ascidia malaca chorion the presence of substances with a polysaccharidic nature that are probably conjugated as glycoprotein and/or proteoglycans.

The test of the Ciona intestinalis larva, before hatching, presents a single outer cuticular layer, C1. Ultrastructural observations of swimming larvae, kept in culture at 18 degrees C, show that the test is enriched by a second inner cuticular layer C2. The C2 layer begins to form in the larva immediately after hatching (about 1 h) and is a process caused by the activity of the cells of the ectodermal layer. During this phase of larval development these cells assume the typical ultrastructure of cells with secretory activity, presenting a well developed r.e.r. and a Golgi in the form of dictyosomes in active synthesis phase in the apical zone of their cytoplasm. The formation of C2 layer is complete in the larva 3 h after hatching and in this period it can be seen parallel to, and a short distance from, the plasmalemma of the ectodermal cells. In the cephalenteron region the C2 layer progressively rises towards the C1 layer and, in the larva 8 h after hatching, these two layers are very close together, about 1000 A apart. In contrast, the C2 layer in the tail remains close to the plasmalemma of the ectodermal cells and can only be detected by ultrastructural observations.