Anna Maria Rinaldi

Cadmium and mitochondria

The heavy metal cadmium (Cd) a pollutant associated with several modern industrial processes, is absorbed in significant quantities from cigarette smoke, water, food and air contaminations. It is known to have numerous undesirable effects on health in both experimental animals and humans, targeting kidney, liver and vascular system. The molecular mechanism accounting for most of the biological effects of Cd are not well-understood and the toxicity targets are largely unidentified. The present review focuses on important recent advances about the effects of cadmium on mitochondria of mammalian cells. Mitochondria are the proverbial powerhouses of the cell, running the fundamental biochemical processes that produce energy from nutrients using oxygen. They are among the key intracellular targets for different stressors including Cd. This review provides new additional informations on the cellular and molecular aspects of the interaction between Cd and cells, emphasizing alterations of mitochondria as important events in Cd cytotoxicity, thus representing an important basis for understanding the mechanisms of cadmium effect on the cells.

Cadmium induces an apoptotic response in sea urchin embryos.

Abstract Cadmium is a heavy metal toxic for living organisms even at low concentrations. It does not have any biological role, and since it is a permanent metal ion, it is accumulated by many organisms. In the present paper we have studied the apoptotic effects of continuous exposure to subacute/sublethal cadmium concentrations on a model system: Paracentrotus lividus embryos. We demonstrated, by atomic absorption spectrometry, that the intracellular amount of metal increased during exposure time. We found, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, that long treatments with cadmium triggered a severe DNA fragmentation. We demonstrated, by immunocytochemistry on whole-mount embryos, that treatment with cadmium causes activation of caspase-3 and cleavage of death substrates α-fodrin and lamin A. Incubating the embryos since fertilization with Z-DEVD FMK, a caspase-3 inhibitor, we found, by immunocytochemistry, that cleavage by caspase-3 and cleavage of death substrates were inactivated.

Confocal microscopy study of the distribution, content and activity of mitochondria during Paracentrotus lividus development

In the present paper we applied confocal microscopy and fluorescence technologies for studying the distribution and the oxidative activity of sea urchin (Paracentrotus lividus) mitochondria during development, by in vivo incubating eggs and embryos with cell‐permeant MitoTracker probes. We calculated, by a mathematical model, the intensity values, the variations of intensity, and the variation index of incorporated fluorochromes. Data demonstrate that mitochondrial mass does not change during development, whereas mitochondrial respiration increases. In addition, starting from 16 blastomeres stage, some regions of the embryo contain organelles more active in oxygen consumption.

Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

Abstract Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution.

Cytoplasmic giant RNA in sea urchin embryos. II. Physicochemical characterization

Abstract It is shown by sucrose gradient analysis, by Sepharose chromatography, and by electrophoresis under denaturing conditions, that the slow electrophoretic mobility of part of the heterogeneous cytoplasmic RNA of sea urchin embryos is actually due to its very high molecular weight.

Cytoplasmic giant RNA in sea urchin embryos. III. Polysomal localization

Abstract It is demonstrated that some heterogeneous RNA including gigantic classes is extractable from polysomes of sea urchin embryos. A major part of this RNA is shifted to lighter zones of the sucrose gradient under conditions that cause polysomal breakdown either in vitro or in vivo. It also is suggested that a substantial part of this RNA contains poly-A sequences.

Centrifugation does not alter spatial distribution of `BEP4' mRNA in paracentrotus lividus EGG

Paracentrotus lividus unfertilized eggs were centrifuged in a sucrose gradient, so to split each into two parts: a nucleated light fragment and an anucleated heavy fragment. Northern blot analyses utilizing a bep4 probe as animal marker and H2A histone gene and 12S‐mit RNA as controls indicate that the eggs are elongated along the animal‐vegetal axis during centrifugation and thereafter split into an animal and a vegetal half. Treatment of the eggs with colchicine before centrifugation abolishes the animal localization of bep4 mRNA.

Hsp56 mRNA in Paracentrotus lividus embryos binds to a mitochondrial protein

We previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is constitutively expressed during development, that it has a specific territorial distribution, both in normal and heat‐shocked embryos, and that its amount increases after heat shock [Roccheri MC, Patti M, Agnello M, Gianguzza F, Carra E, Rinaldi AM. Localization of mitochondrial Hsp56 chaperonin during sea urchin development. Biochem Biophys Res Commun 2001;287:1093–98] and cadmium treatment [Roccheri MC, Agnello M, Boneventura R, Matranga V. Cadmium induces the expression of specific stress proteins in sea urchin embryos. Biochem Biophys Res Commun 2004;321:80–7]. In this study, we looked at Hsp56 mRNA during normal development and under stress conditions. The messenger is almost constantly expressed at all stages of development and its amount is steadily increased in stressed embryos. Moreover, we found, using T1 RNase protection assay, that the most proximal region of the 3′‐UTR of the Hsp56 mRNA binds a 40 kDa protein: this factor is more abundant in the mitochondrial extract and, more specifically, in the outer membrane of the organelle.

MITOCHONDRIA DURING SEA URCHIN OVOGENESIS

Sea urchin represents an ideal model for studies on fertilization and early development, but the achievement of egg competence and mitochondrial behaviour during oogenesis remain to be enlightened. Oocytes of echinoid, such as sea urchin, unlike other echinoderms and other systems, complete meiotic maturation before fertilization. Mitochondria, the powerhouse of eukaryotic cells, contain a multi-copy of the maternally inherited genome, and are involved directly at several levels in the reproductive processes, as their functional status influences the quality of oocytes and contributes to fertilization and embryogenesis. In the present paper, we report our latest data on mitochondrial distribution, content and activity during Paracentrotus lividus oogenesis. The analyses were carried out using confocal microscopy, in vivo incubating oocytes at different maturation stages with specific probes for mitochondria and mtDNA, and by immunodetection of Hsp56, a well known mitochondrial marker. Results show a parallel rise of mitochondrial mass and activity, and, especially in the larger oocytes, close to germinal vesicle (GV) breakdown, a considerable increase in organelle activity around the GV, undoubtedly for an energetic aim. In the mature eggs, mitochondrial activity decreases, in agreement with their basal metabolism. Further and significant information was achieved by studying the mitochondrial chaperonin Hsp56 and mtDNA. Results show a high increase of both Hsp56 and mtDNA. Taken together these results demonstrate that during oogenesis a parallel rise of different mitochondrial parameters, such as mass, activity, Hsp56 and mtDNA occurs, highlighting important tools in the establishment of developmental competence.

Hsp56 protein and mRNA distribution in normal and stressed P.lividus embryos

Abstract It was previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is constitutively expressed during development, that it increases after heat-shock and cadmium treatment, and that it has a specific territorial distribution, both in normal and heat-shocked embryos, as shown by immunolocalization experiments. In this work, we analyzed by Western blot the territorial distribution of the protein in plutei exposed to heat-shock or sublethal cadmium concentrations, and we found that Hsp56 increases in both ectodermal and endodermal cells. Moreover, by “in situ” hybridization, we looked at Hsp56 mRNA during normal development and under stress conditions. We found that the territorial distribution of the messenger changes during development and that its amount is steadily increased in stressed embryos. Finally, by T1 RNase assay, we identified a cytoplasmic factor that binds to the region of Hsp56 messenger containing the 5’UTR.