Sunflower metallothionein family characterisation. Study of the Zn(II)- and Cd(II)-binding abilities of the HaMT1 and HaMT2 isoforms.

Plant metallothioneins (MTs) constitute a family of small Cys-rich proteins capable of coordinating metal ions, significantly differing from microbial and animal MTs. They are divided into four subfamilies depending on the Cys pattern in their sequence. In this work, the MT system of the sunflower plant (Helianthus annuus) has been defined, with ten genes coding for MTs (HaMT) belonging to the four plant MT subfamilies; three HaMT1, four HaMT2, one HaMT3 and two HaMT4 isoforms. The gene expression pattern and capacity to confer metal resistance to yeast cells have been analysed for at least one member of each subfamily. The divalent metal ion-binding abilities of HaMT1-2 and HaMT2-1 (the isoforms encoded by the most abundantly expressed HaMT1 and HaMT2 isogenes) have been characterised, as HaMT3 and HaMT4 were previously studied. Those isoforms constitute an optimum material to study the effect of Cys number variability on their coordination abilities, as they exhibit additional Cys residues regarding the canonical Cys pattern of each subfamily. Our results show that the variation in the number of Cys does not drastically modify their M(II)-binding abilities, but instead modulates the degree of heterogeneity of the corresponding recombinant syntheses. Significantly, the Zn(II)-HaMT1 complexes were highly susceptible to proteolytic cleavage. The recombinant Cd-MT preparations of both isoforms exhibit significant acid-labile sulphide content-Cd6S8 or Cd7S7 species. Overall results suggest that HaMT2-1 is probably associated with Cd(II) detoxification, in contrast to HaMT1-2, which may be more related to physiological functions, such as metal ion transport and delivery.

The response of the different soybean metallothionein isoforms to cadmium intoxication.

Cadmium is a highly toxic heavy metal for both plants and animals. The presence of Cd in agricultural soils is of major concern regarding its entry into the food chain, since Cd compounds are readily taken up by plants, and accumulated in edible parts due to their high solubility. In this study, we first demonstrate the high capacity for Cd concentration of soybean grains. Consequently, we considered the study and characterization of the molecular determinants of Cd accumulation -such as metallothioneins (MT)- to be of major practical importance. We report here the first characterization of the soybean MT system, with the identification of nine genes (one of which is a truncated pseudogene), belonging to the four plant MT types. The most highly expressed of each type was chosen for further function analysis. All of them are expressed at high levels in soybean tissues: GmMT1, GmMT2 and GmMT3 in roots, shoots and seeds, and GmMT4 only in seeds. The corresponding recombinant soybean MTs, synthesized in Escherichia coli cells cultured in metal supplemented media, exhibit greater cadmium than zinc binding capacity. These results suggest a definite role of GmMTs in Cd(II) accumulation as one of the main responses of soybean to an overload of this metal.

Extranodal Rosai-Dorfman disease as a pelvic mass.

Rosai-Dorfman disease (RDD) is a rare histiocytic disorder of unknown cause. RDD most commonly involves the cervical lymph nodes, but extranodal involvement has been described. We report the case of a patient with extranodal RDD that presented as a retroperitoneal mass obstructing the left ureter. The patient underwent surgical resection of the mass, followed by a 5-month course of vinblastine. There was no evidence of progressive disease 22 months after surgery.

Haemodynamic strategy for treatment of diastolic anterograde giacomini varicose veins.

OBJECTIVES: To assess the diagnosis and outcome of a haemodynamic strategy for the treatment of primary varicose veins associated with anterograde diastolic flow (ADF) in the Giacomini vein (GV). METHODS: ADF in the GV, with the escape point located at the saphenopopliteal junction, was demonstrated in 15 patients (15 limbs) by duplex ultrasound. No other escape points were seen in this group. ADF was defined as the flow present in the relaxing phase after isometric contraction of the lower limb, measured in the standing position. Duplex and clinical follow-up was performed prospectively at 1 week, at 1, 3, 6, and 12 months and once per year thereafter, between 1998 and 2001. Surgery consisted of flush division of the GV from the small saphenous vein (SSV) and division of the incompetent collateral veins from the GV. RESULTS: GV diameter showed an average reduction from 6 to 4 mm 33 months after surgery. Fourteen patients (93%) showed no symptoms or varicose veins. GV reconnection and recurrent ADF was demonstrated in two patients (13%). CONCLUSIONS: ADF is a rare condition associated with primary varicose veins. ADF occurs when there is a closed venovenous shunt with recirculation in the muscular diastole. This implies that, although a part of the circuit is ascendant, the re-entry point must be located downstream to the escape point. Accurate duplex assessment is required to distinguish this atypical haemodynamic condition from an abnormal systolic circuit bypassing a deep vein obstruction. Interruption of the GV above its junction with the SSV abolished ADF with an acceptable rate of recurrences.

Durability of reflux-elimination by a minimal invasive CHIVA procedure on patients with varicose veins. A 3-year prospective case study.

OBJECTIVES: to assess the outcome of a conservative and haemodynamic method for insufficient veins on an ambulatory basis (French acronym, "CHIVA") with preservation of the greater saphenous vein (GSV) for treatment of primary varicose veins. METHODS: duplex incompetence of the sapheno-femoral junction (SFJ) and the GSV trunk, with the re-entry perforating point located on a GSV tributary was demonstrated in 58 patients with varices (58 limbs). The re-entry point was defined as the perforator, whose compression of the superficial vein above its opening eliminates reflux in the GSV. Duplex scanning was performed preoperatively and at 7 days, and patients were followed prospectively at 1, 3, 6, 12, 24, and 36 months after CHIVA. Operation consisted in flush ligation and division from the GSV of the tributary containing the re-entry perforating vein (no additional high ligation is included). If reflux returned, SFJ interruption was performed in a second surgical procedure. RESULTS: the GSV diameter showed an average reduction from 6.6 to 3.9 mm 36 months after surgery. Reflux in the GSV system was demonstrated in all but five (8%) patients. Of the 53 patients with recurrent reflux, 46 underwent SFJ interruption. CONCLUSIONS: elimination of reflux in the GSV after the interruption of insufficient collaterals is only temporary.

A new insight into metallothionein (MT) classification and evolution. The in vivo and in vitro metal binding features of Homarus americanus recombinant MT.

We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous Escherichia coli expression in zinc- and copper-supplemented medium was achieved for MTH, the two domains betabetaMTH and betaalphaMTH and three site-directed mutants, betabetaC9H, betaalphaC37H, and betaalphaE31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of M(II)6-MTH for the entire MTH and M(II)3-betabetaMTH and M(II)3-betaalphaMTH for the independent domains fully validated our expression system. A low affinity binding site for a seventh Zn(II) in the in vivo synthesized MTH was located in the betaalpha domain. Additionally, minor M(II)4 species were found for each domain. Both single Cys to His mutations exhibited a similar reduction of their in vivo zinc binding ability but differed in their cadmium binding behavior when compared with the wild-type forms. Conversely, the double mutant showed an enhanced zinc and cadmium binding capacity. In vivo synthesis of MTH and of its independent domains in the presence of copper only afforded heterometallic copper-zinc species. These findings allow consideration of MTH as a zinc thionein and question the view of all crustacea MT structures as copper thioneins. Furthermore, a new approach for the evolutionary and functional classification of MT is proposed, based on the stoichiometry of metal-MT species and molecular phylogenetic analysis.

Zinc(II) is required for the in vivo and in vitro folding of mouse copper metallothionein in two domains.

We postulate that zinc(II) is a keystone in the structure of physiological mouse copper metallothionein 1 (Cu-MT 1). Only when Zn(II) is coordinated does the structure of the in vivo- and in vitro-conformed Cu-MT species consist of two additive domains. Therefore, the functionally active forms of the mammalian Cu-MT may rely upon a two-domain structure. The in vitro behaviour of the whole protein is deduced from the Cu titration of the apo and Zn-containing forms and compared with that of the independent fragments using CD, UV-vis, ESI-MS and ICP-AES. We propose the formation of the following Cu, Zn-MT species during Zn/Cu replacement in Zn7-MT: (Zn4)alpha(Cu4Zn1)beta-MT, (Cu3Zn2)alpha(Cu4Zn1)beta-MT and (Cu4Zn1)alpha(Cu6)beta-MT. The cooperative formation of (Cu3Zn2)alpha(Cu4Zn1)beta-MT from (Zn4)alpha(Cu4Zn1)beta-MT indicates that the preference of Cu(I) for binding to the beta domain is only partial and not absolute, as otherwise accepted. Homometallic Cu-MT species have been obtained either from the apoform of MT or from Zn7-MT after total replacement of zinc. In these species, copper distribution cannot be inferred from the sum of the independent alpha and beta fragments. The in vivo synthesis of the entire MT in Cu-supplemented media has afforded Cu7Zn3-MT [(Cu3Zn2)alpha(Cu4Zn1)beta-MT], while that of alpha MT has rendered a mixture of Cu4Zn1-alpha MT (40%), Cu5Zn1-alpha MT (20%) and Cu7-alpha MT (40%). In the case of beta MT, a mixture of Cu6-beta MT (25%) and Cu7-beta MT (75%) was recovered [1]. These species correspond to some of those conformed in vitro and confirm that Zn(II) is essential for the in vivo folding of Cu-MT in a Cu-rich environment. A final significant issue is that common procedures used to obtain mammalian Cu6-beta MT from native sources may not be adequate.

Drosophila MTN: a metazoan copper-thionein related to fungal forms.

Two Drosophila metallothioneins (MT) have been reported: MTN, a 40 residue peptide including 10 Cys, and MTO, a 43 residue peptide including 12 Cys. However, neither functional nor evolutionary analyses for either of the Drosophila MT are available. Here, heterologous expression of Mtn in Escherichia coli is reported. The metal binding abilities of the Cu- and Zn-MTN complexes conformed in vivo, as well as the features of the Cd- and Cu-aggregates produced by metal replacement in vitro, have been determined by atomic emission spectrometry, circular dichroism and electrospray ionization mass spectrometry. Primary structure relationships with other MT have been examined. The results indicate a close resemblance of MTN to fungal copper-thioneins.

In vivo copper- and cadmium-binding ability of mammalian metallothionein beta domain.

The beta domain of mouse metallothionein 1 (betaMT) was synthesized in Escherichia coli cells grown in the presence of copper or cadmium. Homogenous preparations of Cu-betaMT and Cd-betaMT were used to characterize the corresponding in vivo-conformed metal-clusters, and to compare them with the species obtained in vitro by metal replacement to a canonical Zn3-betaMT structure. The copper-containing betaMT clusters formed inside the cells were very stable. In contrast, the nascent beta peptide, although it showed cadmium binding ability, produced a highly unstable species, whose stoichiometry depended upon culture conditions. The absence of betaMT protein in E. coli protease-proficient hosts grown in cadmium-supplemented medium pointed to drastic proteolysis of a poorly folded beta peptide, somehow enhanced by the presence of cadmium. Possible functional and evolutionary implications of the bioactivity of mammalian betaMT in the presence of monovalent and divalent metal ions are discussed.

A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain.

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.