Isolation and characterization of Mytilus edulis metallothionein genes

Metallothioneins (MTs) are crucial proteins in all organisms for the regulation of essential metals and the detoxification of heavy metals. Many studies have estimated MT levels in mussel tissues to detect marine metal pollution. In this study, we investigated the MT gene structures of the forms present in Mytilus edulis (blue mussel). One MT-10 (2413 bp) gene and one MT-20 (1906 bp) gene were obtained. These MT genes contain three exons and two long introns. The splicing signals for MT-10 and MT-20 were GTA(T/A)GT-(C/T)AG. The structural organization (length of intron, splicing signals, AT content) of MT-10 and MT-20 is compared with other MT genes.     

Nuclear and cytosolic distribution of metallothionein in the blue mussel Mytilus edulis L

Four tissues from the blue mussel, Mytilus edulis L., were examined for the presence of nuclear metallothionein (MT), and the nuclear:cytosolic (N:C) MT ratios and nuclear MT:DNA ratios investigated. Gill, digestive gland, gonad and posterior adductor muscle tissues were dissected, homogenized and subjected to differential centrifugation in order to isolate the nuclear and cytosolic fractions, which were then analyzed for MT and DNA. MT was present in all samples of the nuclear fractions from all four tissues. The nuclear MT concentration was either lower or the same as the cytosolic MT concentration from the same tissue. The mean N:C MT ratio of the digestive gland was significantly lower than that of the gill. The mean nuclear MT:DNA ratio of the digestive gland was significantly higher than that of the gill and posterior adductor muscle. In addition to being the first report of nuclear MT in bivalves, we showed that N:C MT ratios and nuclear MT:DNA ratios differ among tissues of the same organism. This raises important questions concerning the regulation of nuclear MT concentrations and the role of nuclear MT in metal regulation and DNA protection.     

Induction and characterization of metallothionein in chicken epiphyseal growth plate cartilage chondrocytes

Following exposure to cadmium or zinc, chickens were sacrificed and the liver, kidney, and bone epiphyseal growth plates harvested. When cytosolic extracts of the growth plate cartilage were fractionated by gel filtration chromatography, a protein with high metal-binding capacity and low ultraviolet (UV) absorbance eluted in the same position as liver metallothionein (MT) and a MT standard. Cd or Zn treatment resulted in a 25-fold or 5-fold induction in growth plate MT, respectively. In liver the greatest level of MT induction was seen with short-term Cd exposures. In contrast, MT levels in the growth plate increased as the duration of Cd exposure increased. Induction of MT in growth plate chondrocyte cell cultures was observed for media Cd concentrations of ≥0.1 μM and Zn concentrations of ≥100 μM. Basal and inducible levels of MT declined through the culture period and were lowest in the terminally differentiated mineralized late stages of the culture. Alkaline phosphatase activity was also lowest in the late-stage cultures, while total cellular protein increased throughout the culture period. Treatment of chondrocytes with Zn prior to Cd exposure resulted in a protective induction of MT. Pre-treatment of chondrocytes with dexamethasone resulted in suppressed synthesis of MT upon Cd exposure and greater Cd toxicity. Both Cd and Zn resulted in significantly increased levels of MT mRNA in chondrocyte cell cultures. Dexamethasone treatment resulted in an approximate 2- to 3-fold increase in MT mRNA. This is contrary to the finding that MT protein levels were decreased by dexamethasone. The findings suggest that an increased rate of MT degradation in dexamethasone-treated and late-stage chondrocyte cultures may be associated with the terminally differentiated phenotype. J. Cell. Biochem. 68:110–120, 1998. © 1998 Wiley-Liss, Inc.     

Pharmacological Induction of Larval Settlement and Metamorphosis in the Blue Mussel Mytilus edulis L.

The blue mussel Mytilus edulis L. is an important aquaculture and fouling species in northern seas. Although the general role of chemical cues for settlement of larvae of the blue mussel has been proposed, few studies have focused on induction of settlement and metamorphosis by pharmacological agents. In this study, the induction of larval settlement of the blue mussel by pharmacological compounds was investigated through a series of laboratory experiments with an aim of identifying artificial cues for laboratory bioassay systems in fouling and antifouling research. Gamma-aminobutiric acid (GABA), dihydroxyphenyl L-alanine (DOPA), isobutyl methylxanthine (IBMX) and acetylcholine chloride (ACH) at 10^{m 7}-10^{m 2} M as well as KCl at 10-40 mM K⁺ in excess of the level in normal seawater were tested for their inductive effect on larval settlement. In filtered seawater (FSW) <9% of the larvae settled after 48 h. Elevated K⁺ and GABA levels had no effect on larval settlement and metamorphosis. DOPA at 10^{m 5} M and IBMX at 10^{m 6}-10^{m 4} M induced 41-83% larval settlement and ACH at 10^{m 7}-10^{m 5} M induced < 40% larval settlement. While the highest settlement rates were observed after 48 h exposure to the chemicals, most of the larvae settled within 24 h. Compounds at concentrations of 10^{m 3}-10^{m 2} M were either toxic to larvae or retarded the growth of the post-larvae shell. Juveniles resulting from induction by lower concentrations of chemicals had a very high survival rate, completed metamorphosis and grew as well as the juveniles that metamorphosed spontaneously. IBMX at 10^{m 6}-10^{m 4} M and L-DOPA at 10^{m 5} M are effective agents for induction of settlement and metamorphosis for future studies using juvenile M. edulis.     

Turnover rate of metallothionein and cadmium in Mytilus edulis

The results demonstrate the first attempt to determine metallothionein turnover in the whole soft tissues of mussels Mytilus edulis exposed to cadmium. Half-lives for metallothionein and cadmium are 25 and 300 days, respectively. As metallothionein degrades the released cadmium induces further synthesis of the protein, to which the metal becomes resequestered. The slow metallothionein turnover rates (compared with mammals) and the lack of significant cadmium excretion testify to the relatively stable nature of the cadmium-metallothionein complex in these invertebrates and supports the view of a detoxifying role for metallothionein in the mussels.     

Somatic embryogenesis and plant regeneration in different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L⁻¹). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L⁻¹) and NAA (0.1 mg L⁻¹). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L⁻¹). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.     

Prolyl 4-hydroxylase in the foot of the marine mussel Mytilus edulis L.: purification and characterization

The mussel foot secretes a variety of unusual hydroxyproline-containing collagenous and noncollagenous proteins. Prolyl 4-hydroxylase acting on one or more of the secreted proteins was isolated from the foot by using conventional gel filtration and ion exchange chromatography. Mr of the intact enzyme was 230,000 (alpha 2 beta 2) composed of two subunits with Mr of 60,000 (alpha) and 57,000 (beta) as estimated by HPLC gel filtration and SDS-PAGE. The enzyme utilized (Pro-Pro-Gly)10 as a substrate with an apparent Km value of 0.17 mM. Cofactors and inhibitors were very similar to animal, plant, and microbial prolyl hydroxylases previously described. The enzyme had a relatively sharp pH optimum in the range of 7.8-8.3 and the hydroxyproline formed increased in proportion to the rise in the temperature between 5 and 20 degrees C. No detectable hydroxylation occurred with poly-L-proline or the unhydroxylated decapeptide analog (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) of the polyphenolic protein. Kinetic studies, however, revealed that the mussel prolyl 4-hydroxylase was competitively inhibited by poly-L-proline and uncompetitively inhibited by the decapeptide. These results suggest that the decapeptide binds the enzyme-substrate i.e. (Pro-Pro-Gly)10 complex. It is not yet clear whether this enzyme acts exclusively on collagenous substrates or whether its catalytic purview extends as well to the polyphenolic protein.     

Chemical modifications of metallothionein. Preparation and characterization of polymers.

Reaction of rat liver metallothionein-II with two bifunctional cross-linking reagents, glutaraldehyde and dimethyl suberimidate, produces high yields of polymeric species. It is argued that cross-linking is trapping preformed aggregates of the protein, which therefore represent a stabilized quaternary structure of metallothionein. The two polymeric species differ in a number of respects. With dimethyl suberimidate, the polymer retains all metal-binding sites of the monomer, and has an unaltered isoelectric point. Reaction with glutaraldehyde causes loss of one or two Cd2+/Zn2+-binding sites and elevates the pI. Both species are nearly spherical aggregates, in contrast with the highly asymmetrical metallothionein. Both polymers are linked through lysine residues, and the thiol groups remain reduced. The biological significance of these aggregates is discussed.     

Variability of tubule types within the digestive glands of Mercenaria mercenaria (L.), Ostrea edulis L., and Mytilus edulis L.

The variability of the four tubule types previously recognized in the digestive glands of bivalve molluscs (viz. I, holding; II, absorptive; III, fragmenting; and IV, reconstituting) was investigated using photomicrographs of the digestive glands of subtidal Mercenaria mercenaria (L.), low-intertidal Ostrea edulis L., and mid-intertidal Mytilus edulis L. The clustering of similar tubule types around common secondary ducts was observed both histologically and by statistical analysis. A ratio type estimator was used in consideration of this clustering to determine proportions and variances of each tubule type. Intra-animal variances of Type II tubules were approximately the same for each species, ranging from 0.0419–0.0570. Inter-animal variances were also similar (0.0060–0.0140), but only 10–25% of the intraanimal values. Sampling schemes involving large numbers of animals but few photomicrographs of each digestive gland would minimize overall variance. Because of high variability, the necessity of taking numerical data and of using proper statistical analysis in all studies based on the changing morphological appearance of the bivalve digestive gland is stressed.     

Isolation and characterization of a hepatic metallothionein from mice.

Mice were given cadmium chloride orally for 18 weeks, after which they were given two subcutaneous injections of cadmium chloride. Cadmium-and zinc-containing proteins were obtained from livers by ultracentrifugation, Sephadex chromatography and isoelectric focusing. Metallothionein with apI of 4.2 at 8 degrees C was separated from other proteins (possibly including other forms of metallothionein) by the isoelectric procedure. The metallothionein with pI = 4.2 had a high E250, reflecting abundant Cd-SH bonds, in accord with amino acid analysis, which gave a cysteine content of 34.6 residues % and showed an absence of aromatic amino acids. These results indicated a very high purity of the form of metallothionein isolated after isoelectric focusing.     

Induction of metallothionein mRNA in rat liver and kidney after copper chloride injection.

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.     

Localization of metallothionein in female reproductive organs of rat and guinea pig

We demonstrated the localization of metallothionein (MT) in rat uterus and ovaries and in guinea pig mammary glands. During the cyclic changes from one estrous period to the next, strong MT immunostaining was found in the glandular epithelium of the endometrium and weak immunostaining was observed in the simple columnar epithelium. Interestingly, during estrus, the intensity of MT immunostaining decreased in the cytoplasm, whereas during metestrus, diestrus, and proestrus the intensity of strong and similar immunostaining was observed in both the cytoplasm and nucleus. During proestrus and estrus, the number of vaginal epithelial cells containing MT increased on the luminal side of the epithelium and inside the lumen. In rat ovary, strong immunostaining was observed in the cytoplasm and nucleus of granulosa-lutein cells of the corpus luteum and in the cytoplasm of the ovum. In mammary gland of non-pregnant guinea pig, very strong but scattered MT immunostaining was demonstrated in both cytoplasm and nucleus of some epithelial cells of the lactiferous ducts. The mammary tissue of the pregnant guinea pig showed an increase in MT staining in alveolar cells that had proliferated due to pregnancy. The presence of MT in the female reproductive organs, the tissues of which actively grow under the control of female sex hormones, indicates some as yet unknown association of MT with cell proliferation and differentiation.