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Elio Parisi - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning and sequencing of Metallothionein in squamates: new insights into the evolution of the Metallothionein genes in vertebrates.
Gene, 2008Co-Authors: Francesca Trinchella, Marilisa Riggio, Elio Parisi, Silvana Filosa, Rosaria ScudieroAbstract:Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate Metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of Metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian Metallothioneins, we have cloned and sequenced the cDNAs encoding for Metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of Metallothioneins in reptiles. Phylogenetic analysis shows that reptilian Metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified Metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate Metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of Metallothionein genes along the major vertebrate lineages.
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Fish and mammalian Metallothioneins: a comparative study
Gene, 2004Co-Authors: Rosaria Scudiero, Piero Andrea Temussi, Elio ParisiAbstract:Abstract Structural studies show that fish and mammalian Metallothioneins are endowed of distinctive features. In particular, the ninth cysteine residue present in the α domain of fish Metallothionein is shifted of two positions with respect to the mammalian Metallothionein, introducing a conformational modification in the protein structure. In addition, the fish Metallothionein is less hydrophobic and more flexible than its mammalian counterpart. Our previous studies showed that the hydropathy of piscine and mammalian Metallothioneins is significantly correlated with organismal temperature. In the present paper we have performed phylogenetic comparative analysis on Metallothioneins of 24 species of fish and mammals. The results of such analysis failed to indicate that Metallothionein hydropathy is an adaptive response to the thermal regime of the species. We concluded that Metallothionein hydropathy is a trait that did not evolve in association with environmental changes.
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Solution structure of MT_nc, a novel Metallothionein from the Antarctic fish Notothenia coriiceps.
Structure (London England : 1993), 2003Co-Authors: Clemente Capasso, Elio Parisi, Vincenzo Carginale, Orlando Crescenzi, Daniela Di Maro, Roberta Spadaccini, Piero A. TemussiAbstract:Abstract The structure of [ 113 Cd 7 ]-Metallothionein (MT_nc) of the Antarctic fish Notothenia coriiceps , the first three-dimensional structure of a fish Metallothionein, was determined by homonuclear 1 H NMR experiments and heteronuclear [ 1 H, 113 Cd]-correlation spectroscopy. MT_nc is composed of an N-terminal β domain with 9 cysteines and 3 metal ions and a carboxy-terminal α-domain with 11 cysteines and 4 metal ions. The position of the ninth Cys of the α domain of MT_nc is different from the corresponding Cys of mammalian MTs. As a result, the last CXCC motif in the mammalian MT sequence becomes CXXXCC in the fish MT. This difference leads to a structural change of the α domain and, in turn, to a different charge distribution with respect to that observed in mammalian Metallothioneins.
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Stability and conformational dynamics of Metallothioneins from the antarctic fish Notothenia coriiceps and mouse.
Proteins, 2002Co-Authors: Clemente Capasso, Rosaria Scudiero, Elio Parisi, Vincenzo Carginale, Omoefe Abugo, Fabio Tanfani, Andrea Scirè, Sabato D'auriaAbstract:The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse Metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two Metallothioneins is similar to that of other Metallothioneins, most of which lack periodical secondary structure elements such as α-helices and β-sheets. However, the infrared spectra of the N. coriiceps Metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to α-helices whose content resulted in 5% of the total secondary structure of the protein. The short α-helix found in N. coriiceps Metallothionein showed an onset of denaturation at 30°C and a Tm at 48°C. The data suggest that in N. coriiceps Metallothionein a particular cysteine is involved in the α-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps Metallothionein possesses a more flexible structure than mouse Metallothionein. The spectroscopic data are discussed in terms of the biological function of the Metallothioneins. Proteins 2002;46:259–267. © 2002 Wiley-Liss, Inc.
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Metallothionein in Antarctic notothenioids: Genetic polymorphism and differential gene expression
Italian Journal of Zoology, 2000Co-Authors: Vincenzo Carginale, Rosaria Scudiero, Clemente Capasso, Elio ParisiAbstract:Abstract Among Antarctic notothenioids, appreciable amounts of hepatic Metallothionein were found in red‐blooded species, whilst very little Metallothionein, if any, was detected in icefish. In contrast, high levels of Metallothionein mRNA were revealed in both fishes by Northern blot analysis of total RNA. Phylogenetic analyses carried out on the Metallothionein sequences obtained by using reverse‐transcriptase polymerase chain reaction on eight notothenioid species revealed that notothenioid Metallothioneins can be divided into two groups, Metallothionein‐I and Metallothionein‐II, indicating that they arose from at least one gene duplication event occurring in the ancestral lineage of the Notothenioidei. A strong induction of Metallothionein gene expression and Metallothionein accumulation could be obtained in icefish injected with cadmium salt. Apparently, the expression of the genes encoding MT‐I and MT‐II in icefish liver is differentially regulated by cadmium. The discrepancy between Metallothionein...
Peter James - One of the best experts on this subject based on the ideXlab platform.
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purification and primary structure of Metallothioneins induced by cadmium in the protists tetrahymena pigmentosa and tetrahymena pyriformis
FEBS Journal, 1994Co-Authors: Ester Piccinni, Werner Staudenmann, Vincenzo Albergoni, Raffaele De Gabrieli, Peter JamesAbstract:Tetrahymena pyriformis and Tetrahymena pigmentosa grown in the presence of a non-toxic dose of cadmium, accumulate the metal in the cytosol. Purification by gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography showed that the metal is bound principally to newly formed proteins with ultraviolet spectra and cysteine contents similar to those of Cd2+-Metallothioneins from multicellular organisms. The isolated proteins revealed that the two species of ciliates each express two Cd2+-isothioneins. The primary structures determined by both Edman degradation and mass spectrometry revealed that the equivalent proteins from T. pyriformis and T. pigmentosa have identical sequences and that the two isoforms in each species differ only by the presence or absence of a lysine residue at the N-terminus. The development of automated mass spectrometric sequence analysis algorithms combined with an accurate determination of the molecular mass allowed the rapid confirmation of the sequences. The Tetrahymena Metallothionein sequences are unusually long (105 and 104 amino acids) and show a unique internal homology which suggests that the proteins arose by gene duplication. The chains contain 31 cysteine residues, 15 of which are arranged in motifs characteristic of the mammalian Metallothioneins; the remaining residues show several unique repeating motifs, which could have interesting consequences for the tertiary structure of the metal-binding sites. Amino acid sequences of Tetrahymena Metallothioneins have some similarity with other eukaryotic Metallothioneins. A comparison on the basis of optimised FASTA scores, shows a closer relationship with horse Metallothionein-1B.
Ester Piccinni - One of the best experts on this subject based on the ideXlab platform.
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purification and primary structure of Metallothioneins induced by cadmium in the protists tetrahymena pigmentosa and tetrahymena pyriformis
FEBS Journal, 1994Co-Authors: Ester Piccinni, Werner Staudenmann, Vincenzo Albergoni, Raffaele De Gabrieli, Peter JamesAbstract:Tetrahymena pyriformis and Tetrahymena pigmentosa grown in the presence of a non-toxic dose of cadmium, accumulate the metal in the cytosol. Purification by gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography showed that the metal is bound principally to newly formed proteins with ultraviolet spectra and cysteine contents similar to those of Cd2+-Metallothioneins from multicellular organisms. The isolated proteins revealed that the two species of ciliates each express two Cd2+-isothioneins. The primary structures determined by both Edman degradation and mass spectrometry revealed that the equivalent proteins from T. pyriformis and T. pigmentosa have identical sequences and that the two isoforms in each species differ only by the presence or absence of a lysine residue at the N-terminus. The development of automated mass spectrometric sequence analysis algorithms combined with an accurate determination of the molecular mass allowed the rapid confirmation of the sequences. The Tetrahymena Metallothionein sequences are unusually long (105 and 104 amino acids) and show a unique internal homology which suggests that the proteins arose by gene duplication. The chains contain 31 cysteine residues, 15 of which are arranged in motifs characteristic of the mammalian Metallothioneins; the remaining residues show several unique repeating motifs, which could have interesting consequences for the tertiary structure of the metal-binding sites. Amino acid sequences of Tetrahymena Metallothioneins have some similarity with other eukaryotic Metallothioneins. A comparison on the basis of optimised FASTA scores, shows a closer relationship with horse Metallothionein-1B.
Rosaria Scudiero - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning and sequencing of Metallothionein in squamates: new insights into the evolution of the Metallothionein genes in vertebrates.
Gene, 2008Co-Authors: Francesca Trinchella, Marilisa Riggio, Elio Parisi, Silvana Filosa, Rosaria ScudieroAbstract:Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate Metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of Metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian Metallothioneins, we have cloned and sequenced the cDNAs encoding for Metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of Metallothioneins in reptiles. Phylogenetic analysis shows that reptilian Metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified Metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate Metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of Metallothionein genes along the major vertebrate lineages.
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Fish and mammalian Metallothioneins: a comparative study
Gene, 2004Co-Authors: Rosaria Scudiero, Piero Andrea Temussi, Elio ParisiAbstract:Abstract Structural studies show that fish and mammalian Metallothioneins are endowed of distinctive features. In particular, the ninth cysteine residue present in the α domain of fish Metallothionein is shifted of two positions with respect to the mammalian Metallothionein, introducing a conformational modification in the protein structure. In addition, the fish Metallothionein is less hydrophobic and more flexible than its mammalian counterpart. Our previous studies showed that the hydropathy of piscine and mammalian Metallothioneins is significantly correlated with organismal temperature. In the present paper we have performed phylogenetic comparative analysis on Metallothioneins of 24 species of fish and mammals. The results of such analysis failed to indicate that Metallothionein hydropathy is an adaptive response to the thermal regime of the species. We concluded that Metallothionein hydropathy is a trait that did not evolve in association with environmental changes.
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Stability and conformational dynamics of Metallothioneins from the antarctic fish Notothenia coriiceps and mouse.
Proteins, 2002Co-Authors: Clemente Capasso, Rosaria Scudiero, Elio Parisi, Vincenzo Carginale, Omoefe Abugo, Fabio Tanfani, Andrea Scirè, Sabato D'auriaAbstract:The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse Metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two Metallothioneins is similar to that of other Metallothioneins, most of which lack periodical secondary structure elements such as α-helices and β-sheets. However, the infrared spectra of the N. coriiceps Metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to α-helices whose content resulted in 5% of the total secondary structure of the protein. The short α-helix found in N. coriiceps Metallothionein showed an onset of denaturation at 30°C and a Tm at 48°C. The data suggest that in N. coriiceps Metallothionein a particular cysteine is involved in the α-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps Metallothionein possesses a more flexible structure than mouse Metallothionein. The spectroscopic data are discussed in terms of the biological function of the Metallothioneins. Proteins 2002;46:259–267. © 2002 Wiley-Liss, Inc.
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Metallothionein in Antarctic notothenioids: Genetic polymorphism and differential gene expression
Italian Journal of Zoology, 2000Co-Authors: Vincenzo Carginale, Rosaria Scudiero, Clemente Capasso, Elio ParisiAbstract:Abstract Among Antarctic notothenioids, appreciable amounts of hepatic Metallothionein were found in red‐blooded species, whilst very little Metallothionein, if any, was detected in icefish. In contrast, high levels of Metallothionein mRNA were revealed in both fishes by Northern blot analysis of total RNA. Phylogenetic analyses carried out on the Metallothionein sequences obtained by using reverse‐transcriptase polymerase chain reaction on eight notothenioid species revealed that notothenioid Metallothioneins can be divided into two groups, Metallothionein‐I and Metallothionein‐II, indicating that they arose from at least one gene duplication event occurring in the ancestral lineage of the Notothenioidei. A strong induction of Metallothionein gene expression and Metallothionein accumulation could be obtained in icefish injected with cadmium salt. Apparently, the expression of the genes encoding MT‐I and MT‐II in icefish liver is differentially regulated by cadmium. The discrepancy between Metallothionein...
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PCR amplification and cloning of Metallothionein complementary DNAs in temperate and Antarctic sea urchin characterized by a large difference in egg Metallothionein content.
Cellular and molecular life sciences : CMLS, 1997Co-Authors: Rosaria Scudiero, Clemente Capasso, Antonio Capasso, Vincenzo Carginale, Marilisa Riggio, M. Ciaramella, Silvana Filosa, Elio ParisiAbstract:Metallothionein levels were determined in the eggs of two sea urchin species, the Mediterranean Sphaerechinus granularis and the Antarctic Sterechinus neumayeri. While appreciable levels of Metallothionein were found in S. granularis eggs, a negligible amount was detected in S. neumayeri. Two Metallothionein isoforms were purified from S. granularis, and Metallothionein cDNAs were obtained by means of reverse transcriptase-polymerase chain reaction (RT-PCR). Two distinct cDNA species were cloned and sequenced. The translated amino acid sequences of these two forms consisted of 67 residues and differed in two amino acid substitutions. Despite the lack of Metallothionein in S. neumayeri eggs, a Metallothionein cDNA was obtained by RT-PCR amplification and a single amino acid sequence coding for a 63 residues MT was deduced. A comparative analysis of the primary structure of S. granularis and S. neumayeri Metallothioneins with those of the other sea urchin Metallothioneins has been performed. Sea urchin Metallothioneins appear to be less similar to each other than Metallothioneins of closely related vertebrates.
Ryszard Lobinski - One of the best experts on this subject based on the ideXlab platform.
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Probing metal-complexes with Metallothioneins by reversed phase microbore chromatography and capillary zone electrophoresis coupled with inductively coupled plasma and electrospray mass spectrometry.
Cellular and Molecular Biology, 2000Co-Authors: K. Połeć, H. Chassaigne, Sandra Mounicou, Ryszard LobinskiAbstract:Four different hyphenated techniques: microbore reversed phase (RP) HPLC-ICP MS, CZE-ICP MS, RP HPLC-ES MS and CZE-ES MS were investigated for the characterization of Metallothionein-metal complexes under neutral pH conditions. Particular attention was given to the differentiation between Metallothionein and artifact signals, identification of mixed-metal complexes, and the validity of the molecular mass as the identification parameter of the different MT iso- and sub-isoforms. Despite the similar morphology of chromatograms and electrophoregrams mass spectrometry revealed different origin of the apparently corresponding peaks. The performance of the four above mentioned techniques was characterized using the example of rabbit liver MT-1 preparation. Reversed-phase HPLC with post-column acidification prior to ES MS was judged to be the most versatile technique for the characterization of metal complexes with Metallothioneins but other techniques offer valuable auxiliary information.
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Analysis for Metallothioneins using coupled techniques
Talanta, 1998Co-Authors: Ryszard Lobinski, H. Chassaigne, Joanna SzpunarAbstract:Analytical chemistry of Metallothioneins based on the coupling of a high resolution separation technique with an element or species selective detection technique is discussed. The role of size-exclusion chromatography (SEC) with on-line atomic spectrometric detection for the quantification of Metallothionein fraction in cell cytosols is evaluated. Particular attention is given to the conditions for the separation of metallated Metallothionein isoforms (MT-1, MT-2, MT-3) and sub-isoforms within these classes by anion-exchange and reversed-phase HPLC. Techniques for interfacing chromatography with atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP AES) and ICP mass spectrometry (MS) are assessed. The potential of electrospray (tandem) mass spectrometry for the characterization of Metallothionein isoforms with respect to molecular mass and aminoacid sequence is highlighted. Perspectives for capillary zone electrophoresis (CZE), microbore and capillary HPLC with ICP MS and electrospray MS(/MS) detection for the probing of Metallothioneins are discussed. Applications of hyphenated techniques to the analysis of real-world samples are reviewed.
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Characterization of Metallothionein isoforms by reversed-phase high-performance liquid chromatography with on-line post-column acidification and electrospray mass spectrometric detection
J. Chromatogr. A, 1998Co-Authors: H. Chassaigne, Ryszard LobinskiAbstract:Post-column acidification of the chromatographic effluent was developed to eliminate artefacts in investigations of the polymorphism of Metallothionein (MT) by microbore reversed-phase HPLC with detection by pneumatically assisted electrospray mass spectrometry. Metallated species (Cd, Zn and mixed Cd-Zn complexes) were decomposed on-line to produce apo Metallothioneins of which the molecular masses were determined by MS. Besides the simplification of the mass spectra taken at the apexes of the chromatographic peaks, the method resulted in a 10-fold improvement of the detection limit of Metallothionein and allowed a more comprehensive and less ambiguous detection and identification of the iso- and subisoforms. The method was applied to the characterization of rabbit liver Metallothioneins: rabbit liver MT (purified by size-exclusion chromatography only) and MT-1 and MT-2 isoform fractions purified additionally by anion-exchange chromatography. Copyright (C) 1998 Elsevier Science B.V.