Francesco Boldrin

Metal interaction and regulation of Tetrahymena pigmentosa metallothionein genes.

The patterns of expression of two metallothionein (MT) genes, MT-1 and MT-2, previously identified as Cd-MT and Cu-MT, were analysed in Tetrahymena pigmentosa in response to metal inducers cadmium, copper and zinc and to a mixture of copper and cadmium at appropriate concentrations. Co-treatment induces synergistic accumulation of both metals and higher expression of MT-mRNAs in the first few hours. mRNA levels were observed not to completely correlate with MT-protein levels, suggesting that post-transcriptional regulation may be involved in MT induction. MT-1 is induced to higher levels than MT-2. Zinc does not induce any MT expression. The lowest level of mRNA was observed for MT-2, induced only by copper. Cadmium is a powerful inducer of the MT-1 gene, although a very low transcription rate by copper occurs in the first hour.

Metallothionein Gene from Tetrahymena thermophila with a Copper-Inducible-Repressible Promoter

ABSTRACT We describe a novel metallothionein gene from Tetrahymena thermophila that has a strong copper-inducible promoter. This promoter can be turned on and off rapidly, making it a useful system for induction of ectopic gene expression in Tetrahymena and enhancing its applications in cell and molecular biology, as well as biotechnology.

Identification, cloning and characterisation of a novel copper-metallothionein in tetrahymena pigmentosa. Sequencing of cDNA and expression.

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.

CiMT-1, an unusual chordate metallothionein gene in Ciona intestinalis genome: structure and expression studies

The present article reports on the characterization of the urochordate metallothionein (MT) gene, CiMT-1, from the solitary ascidian Ciona intestinalis. The predicted protein is shorter than other known deuterostome MTs, having only 39 amino acids. The gene has the same tripartite structure as vertebrate MTs, with some features resembling those of echinoderm MTs. The promoter region shows the canonical cis-acting elements recognized by transcription factors that respond to metal, ROS, and cytokines. Unusual sequences, described in fish and echinoderms, are also present. In situ hybridization suggests that only a population of hemocytes involved in immune responses, i.e. granular amebocytes, express CiMT-1 mRNA. These observations support the idea that urochordates perform detoxification through hemocytes, and that MTs may play important roles in inflammatory humoral responses in tunicates. The reported data offer new clues for better understanding the evolution of these multivalent proteins from non-vertebrate to vertebrate chordates and reinforce their functions in detoxification and immunity.

Cu,Zn Superoxide Dismutases from Tetrahymena thermophila: Molecular Evolution and Gene Expression of the First Line of Antioxidant Defenses

In the present study, we describe the molecular and functional characterization of two Cu,Zn superoxide dismutase (SOD) genes, named tt-sod1a and tt-sod1b from Tetrahymena thermophila, a free-living ciliated protozoan widely used as model organism in biological research. The cDNAs and the putative amino acid sequences were compared with Cu,Zn SODs from other Alveolata. The primary sequences of T. thermophila Cu,Zn SODs are unusually long if compared to orthologous proteins, but the catalytically important residues are almost fully conserved. Both phylogenetic and preliminary homology modeling analyses provide some indications about the evolutionary relationships between the Cu,Zn SODs of Tetrahymena and the Alveolata orthologous enzymes. Copper-dependent regulation of Cu,Zn SODs expression was investigated by measuring mRNA accumulation and enzyme activity in response to chronic exposure to non-toxic doses of the metal. Our in silico analyses of the tt-sod1a and tt-sod1b promoter regions revealed putative consensus sequences similar to half Antioxidant Responsive Elements (hARE), suggesting that the transcription of these genes directly depends on ROS formation. These data emphasize the importance of complex metal regulation of tt-sod1a and tt-sod1b activation, as components of an efficient detoxification pathway allowing the survival of T. thermophila in continued, elevated presence of metals in the environment.

Comparative study on metal homeostasis and detoxification in two Antarctic teleosts.

The main characteristic of Antarctic seawater is its low constant temperature and its high concentration of O(2), which can increase the formation rate of reactive oxygen species (ROS), together with a natural occurrence of elevated cadmium and copper levels. In the present paper, we studied the presence of cadmium, copper and zinc, metallothioneins (MTs) and glutathione (GSH), and antioxidant enzyme activities in the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi, in order to determine the influence of the peculiar physico-chemical features of the Antarctic marine environment on these physiological defence systems in two species of teleosts. In both of them, cadmium and copper accumulation occurs mostly in the liver. T. bernacchii accumulates zinc mostly in the hepatic tissue, whereas T. newnesi does not show a preferential accumulation site. In addition to the intra-specific analysis, we decided to compare the two species of the Trematomus genus in order to verify if the different feeding habits and motility of these fish affects metal accumulation. Our results show that the liver of T. bernacchii accumulates cadmium and zinc at a higher extent with respect to T. newnesi. Glutathione (GSH) and metallothioneins (MTs) are present in great quantity in the liver of both species. Moreover liver is the tissue which generally showed the highest antioxidant enzyme levels. The results provide further insights in the physiological mechanisms evolved by animals living in this extreme environment.

Metal and metallothionein distribution in different tissues of the Mediterranean clam Venerupis philippinarum during copper treatment and detoxification

Filter feeding animals can accumulate large amount of contaminants in their body through particles filtered from seawater. In particular, copper is interesting since it plays important roles as co-factor of numerous proteins but its toxicity is well established, also because it can readily generate free radicals or oxidize cellular components through their redox activity. Its availability is tightly regulated within cells: it is immediately transferred to metallothionein (MT) that in turn provides efficient and specific mechanisms for its intracellular storage and transport. The aim of this study was to evaluate the acute effect of sublethal copper concentrations in Venerupis philippinarum, by studying the kinetics of copper, zinc (for its interactions at the sites of intake or elimination with the accumulation of other essential and not essential trace metals) and metallothionein accumulation under laboratory conditions. The time-course of metal accumulation/elimination is similar in digestive gland and gills and importantly it is dose-dependent. Both copper and zinc increase slowly within cells, reaching a maximum concentration at the end of the exposure period. During the detoxification period, the metal levels in digestive gland and gills rapidly decrease, with different kinetics in the two tissues. Positive correlations between metallothionein accumulation and copper or zinc concentrations have been verified in both treated groups. The obtained data demonstrated the involvement of MTs in detoxification strategies after a recovery period in clean seawater.

The “Complex” Ascidiosperm of Aplousobranchs

The ascidiosperm of non-appendicularian tunicates has a basic plane: the “head” contains a rod-like nucleus flanked by a large mitochondrion; the acrosome is reduced to small vesicles; the “tail” has a 9+2 axoneme. In species with external fertilisation, the sperm is simpler than in internally fertilising species. Our data show that, in Trididemnum cereum, the long nucleus coils apically in a flange of 40 spires and is flanked by mitochondrion and vesicles; the flagellum has 9 accessory bodies. Other aplousobranchs, the polycitorid Polycitor adriaticus and the polyclinid Aplidium conicum, also have a long nucleus coiled apically in 8 or 6 spires, respectively. Aplidium conicum has a “dense groove” joining the plasma membrane to the nuclear envelope. Although sperm complexity may depend on the mode of fertilisation, it is possible to distinguish plesiomorphies and apomorphies in sperm and use them for the phylogenetic reconstruction of tunicates.

Antidepressants act by inducing autophagy controlled by sphingomyelin–ceramide

Major depressive disorder (MDD) is a common and severe disease characterized by mood changes, somatic alterations, and often suicide. MDD is treated with antidepressants, but the molecular mechanism of their action is unknown. We found that widely used antidepressants such as amitriptyline and fluoxetine induce autophagy in hippocampal neurons via the slow accumulation of sphingomyelin in lysosomes and Golgi membranes and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A and thereby the autophagy proteins Ulk, Beclin, Vps34/Phosphatidylinositol 3-kinase, p62, and Lc3B. Although treatment with amitriptyline or fluoxetine requires at least 12 days to achieve sphingomyelin accumulation and the subsequent biochemical and cellular changes, direct inhibition of sphingomyelin synthases with tricyclodecan-9-yl-xanthogenate (D609) results in rapid (within 3 days) accumulation of ceramide in the ER, activation of autophagy, and reversal of biochemical and behavioral signs of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioral changes typical of MDD. These findings identify sphingolipid-controlled autophagy as an important target for antidepressive treatment methods and provide a rationale for the development of novel antidepressants that act within a few days.