Myoendothelial junctions in blood vessels of a teleost (Prochilodus scrofa) pancreas and caeca

Summary Close contacts between endothelial and smooth muscle cells in teleost (Prochilodus scrofa) blood vessels are described for the first time in the present study. More frequently are seen finger-like, club-shaped or foot-like endothelial processes that come into close contact with the plasma membrane of a smooth muscle cell. Rarely, some myoendothelial contacts occur between the finger-like protrusions that arise from both the endothelial and from the smooth muscle cells. The functional significance of the myoendothelial connection is discussed.     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

Arrested development in Fucus spiralis (Phaeophyceae) germlings exposed to copper

Exposure of Fucus spiralis germlings to precise copper concentrations (0 to 844?nM?Cu²⁺) in chemically defined medium demonstrated a relationship between ultrastructural changes and growth retardation with increasing copper concentration. Electron-translucent vesicles, present in ova, which normally disappear after fertilization, accumulated in germlings exposed to Cu²⁺ above 10.6?nM, suggesting that copper may inhibit a metabolic pathway involved in cell wall formation which is initiated by fertilization. No membrane damage was observed during the exposure period. During a post-exposure period in copper-free medium, recovery occurred (rhizoid extension, apical hair formation) in germlings previously exposed to concentrations below 106?nM?Cu²⁺ and electron-translucent vesicles became granular and disappeared. It is proposed that the electron-translucent vesicles contain a cell wall precursor and that copper inhibits its incorporation into the cell wall, preventing growth and development of the zygote.     

Intracellular pH regulation in maize root tips exposed to ammonium at high external pH

Ammonium-induced changes in the cytoplasmic and vacuolar pH values of excised maize (Zea mays L.) root tips, measured by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy, were correlated with the ammonium content of the tissue, determined by 14N NMR. Calculations based on these measurements indicated that the pH changes observed during exposure to 10 mM ammonium for 1 h at pH 9.0, and in the recovery following the removal of the external ammonium supply, were largely determined by the influx and efflux of the weak base NH3. Carboxylate synthesis, detected by both in vivo 13C NMR and the incorporation of [14C]bicarbonate, was stimulated by the ammonium-induced alkalinization of the root tips, but the contribution that this proton-generating process made to pH regulation during and after the ammonium treatment was quantitatively insignificant. Similarly, ammonium assimilation, which was shown to occur via the proton-generating glutamine synthetase/glutamate synthase pathway using in vivo 15N NMR, was also quantitatively insignificant in comparison with the large changes in ammonium content that occurred during the ammonium treatment and subsequent recovery. The results are discussed in relation to several recent studies in which ammonium was used to perturb intracellular pH values, and it is argued (i) that a new method for probing the subcellular compartmentation of amino acids, based on an ammonium-induced alkalinization of the cytoplasm may be difficult to implement in dense heterogeneous tissues; and (ii) that observations on the apparently proton-consuming effect of ammonium assimilation in rice root hairs may actually reflect unusually rapid assimilation.     

Effects of acute heat exposure on respiratory metabolism and blood glucose in freshwater fishes, Prochilodus scrofa (curimbatá) and Cyprinus carpio (carp) acclimatized to tropical winter

Routine oxygen consumption and blood glucose were determined from freshwater fishes, Prochilodus scrofa and Cyprinus carpio, exposed at high temperatures for 1 hr. Prochilodus scrofa had a significantly higher rate of oxygen consumption at 30 degrees C than at 25 degrees C, and carp higher at 25 degrees C than at 30 degrees C. Blood glucose was significantly higher for Cyprinus carpio than for Prochilodus scrofa at 25 and 30 degrees C; however, after exposure to these temperatures for 1 hr blood glucose did not change significantly for both species. The results suggest that these interspecific variations may be linked to the differences between native and foreign fishes and their way of life.     

Hepatic metallothionein production and resistance to heavy metals by rainbow trout (Salmo gairdneri)--I. Exposed to an artificial mixture of zinc, copper and cadmium

Rainbow trout developed elevated hepatic metallothionein concentrations after 4 weeks in a solution containing zinc, copper and cadmium in a fixed ratio of 400:20:1. Resistance to a combination of these metals increased in proportion to the concentration to which they were exposed for 4 weeks. The concentration of copper but not zinc or cadmium in the low molecular weight proteins separated by gel filtration was related to the concentrations of metallothionein present. The combined toxicity of the metals in the mixture was additive.     

Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.     

Postnatal changes in subcellular distribution of copper,zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation

1. Dramatic interdependent changes in the intracellular concentrations of copper (Cu), zinc (Zn) and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were observed.2. The post-mitochondrial Cu, Zn and MT (ZnMT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (CuMT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex CuMT was translocated to the nuclei.3. The nuclear Cu concentration reached the highest level (62–71% of the total tissue Cu) in the period from day 3 to day 20 post-partum, just prior to and during a rapidly growing liver.4. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.     

Antioxidant and lipid peroxidation responses in Mytilus galloprovincialis exposed to mixtures of benzo(a)pyrene and copper

This study aimed to assess the antioxidant system potential and lipid peroxidative effects, in the gill and digestive gland of Mytilus galloprovincialis exposed to individual and binary mixtures of benzo(a)pyrene (BaP) and Cu for 7 days. Data demonstrated that in mussels exposed to BaP antioxidant enzymes (catalase--CAT, total glutathione peroxidase--tGPx, glutathione S-transferase--GST and glutathione reductase--GR) and lipid peroxidation (LPO) increased in the gill. On the contrary, in the digestive gland inhibitory antioxidant effects (superoxide dismutase-SOD, GR, metallothioneins-MT) and no changes in LPO levels were detected. Cu was also a potent oxidant agent since MT and LPO levels increased in mussel gill, despite no LPO effect in the digestive gland. For both single contaminants the organ specificity and distinct physiologic/metabolism roles were evident in terms of antioxidant capacity. Gill SOD inhibition, MT and GST unchanged was a result of "simple independent action" of exposure to BaP and Cu. "Interactions" in the binary mixtures, led to absence of changes in LPO effects. In the digestive gland, BaP and Cu interactions were also responsible for the GST and LPO enhancement (antagonistic effects). The current findings demonstrate the differences in antioxidant responses where the organ dependency highlights each contaminant particular mode of action. Generally, in the gill "non-interactive" effects occurred with the lowest Cu concentration while "interactions" exist for the mixture with the highest Cu concentrations. In the digestive gland, "interactions" and "no interaction" effects occurred in all the binary mixtures. Complex contaminant mixtures interact differently based on target tissue which may lead to an imbalance in the mussels health status.     

The effect of pH and lysosomal extract on rat liver metallothionein (Cd-thionein)

Was studied in vitro. The¹⁰⁹Cd-labeled protein was isolated by gel filtration and incubated with a lysosomal extract from the same source. No degradation was seen when the pH of the incubation medium was 5 or higher, whereas the degradation of Cd-thionein was completed in 2 h at a pH of 4.5. Dissociation of Cd²⁺ from thionein, which takes place to a fair extent at a pH below 5, appears necessary before the enzymatic breakdown of the thionein moiety can take place.     

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 °C for 4 h under acid conditions, it rapidly declines when heating at a pH above 6.5. To clarify the structural difference at high pH, the atomic structure of a recombinant thaumatin I at pH 8.0 was determined at a resolution of 1.0 Å. Comparison to the crystal structure of thaumatin at pH 7.3 and 7.0 revealed the root-mean square deviation value of a Cα atom to be substantially greater in the large disulfide-rich region of domain II, especially residues 154–164, suggesting that a loop region in domain II to be affected by solvent conditions. Furthermore, B-factors of Lys137, Lys163, and Lys187 were significantly affected by pH change, suggesting that a striking increase in the mobility of these lysine residues, which could facilitate a reaction with a free sulfhydryl residue produced via the β-elimination of disulfide bonds by heating at a pH above 7.0. The increase in mobility of lysine residues as well as a loop region in domain II might play an important role in the heat-induced aggregation of thaumatin above pH 7.0.     

Hepatic aryl hydrocarbon hydroxylase activities in coho salmon (Oncorhynchus kisutch) exposed to petroleum hydrocarbons

Coho salmon exposed to the water soluble fraction (WSF) of Cook Inlet crude oil for a maximum of 30 days showed a greater than three-fold increase in hepatic aryl hydrocarbon hydroxylase activity. The initial increase in enzyme activity appeared between 2 and 5 days of exposure and increased as a function of increased exposure time. Persistence of the induced enzyme activity was dependent on the length and the concentration of WSF exposure. Handling stress had no effect on the AHH activity, but starvation caused a decrease in the activity.     

Physiological responses and expression of metallothionein (MT) and superoxide dismutase (SOD) mRNAs in olive flounder, Paralichthys olivaceus exposed to benzo[a]pyrene

We cloned complementary DNA (cDNA) encoding metallothionein (MT) and superoxide dismutase (SOD) from the liver of olive flounder, Paralichthys olivaceus. The full-length MT cDNA consists of 183 base pairs (bp) and encodes a protein of 60 amino acids; partial SOD cDNA consists of 326 bp and encodes a protein of 109 amino acids. We investigated the dose- and time-related effects of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) on MT and SOD mRNA using quantitative polymerase chain reaction (QPCR). The expression levels of MT mRNA were highest at 24 h (about five times) in 10 microg/L BaP, and at 6 h (about twelve times) in 30 microg/L BaP. The expression levels of SOD mRNA were highest at 12 h (about three times) in 10 microg/L BaP, and at 6 h (about six times) in 30 microg/L BaP, and then decreased toward the end of the experiment. We also measured plasma glucose and cortisol, all of which increased with BaP exposure. These results suggest that MT and SOD play an important role in the detoxification of reactive oxygen species (ROS) caused by BaP exposure, and thus may be indicators of oxidative stress responses.     

Noncooperative cadmium(II) binding to human metallothionein 1a

The two-domain (βα) mammalian metallothionein binds seven divalent metals, however, the binding mechanism is not well characterized and recent reports require the presence of the partially metallated protein. In this paper, step-wise metallation of the metal-free, two-domain βα-rhMT and the isolated β-rhMT using Cd(II) is shown to proceed in a noncooperative manner by analysis of electrospray ionization mass spectrometric data. Under limiting amounts of Cd(II), all intermediate metallation states up to the fully metallated Cd₃-β-rhMT and Cd₇-βα-rhMT were observed. Addition of excess Cd(II), resulted in formation of the supermetallated (metallation in excess of normal levels) Cd₄-β- and Cd₈-βα-metallothionein species. These data establish that noncooperative cadmium metallation is a property of each isolated domain and the complete two-domain protein. Our data now also establish that supermetallation is a property that may provide information about the mechanism of metal transfer to other proteins.     

Location of copper in larvae of Plectrocnemia conspersa (Curtis) (Trichoptera) exposed to elevated metal concentrations in a mine drainage stream

Using histological techniques together with transmission electron microscopy and X-ray microanalysis, copper-containing granules (which also contain significant amounts of sulphur) have been observed in the cells of the malpighian tubules and in the subcuticular region of larvae of Plectrocnemia conspersa. It is suggested that the granules in both these regions are primarily pigment granules, which provide a mechanism for taking potentially toxic concentrations of copper (and possibly other metals) out of circulation. This mechanism of metal tolerance may in part account for the reported occurrence of larvae of P. conspersa in waters with elevated concentrations of trace metals.     

Purification and characterization of metallothionein from liver of cadmium exposed Rhesus monkeys (Macaca mulatta)

Metallothionein (MT) a low molecular weight, Cd-binding, cysteine rich, cytosolic protein has been isolated, purified and characterized from cadmium exposed Rhesus monkeys maintained on protein calorie malnourished (PCM) diet. Metallothionein was resolved into three isoforms i.e. MT_a, MT_b and MTV_c. The ratio of Cd, Zn and Cu varied in these isometallothioneins. MT_c was the major isometallothionein. UV Spectra of MT_c revealed the presence of mercaptide bonds and absence of aromatic amino acids. These observations were further confirmed by amino acid analysis of MT_c which demonstrated high cysteine content (22.6) followed by serine, glycine and lysine. The molecular weight of MT_c as determined by gel filtration and amino acid analysis was 13000 and 6398 daltons respectively. This demonstrates that MT_c is a non-globular ellipsoid polypeptide. MT_c showed a unique property of binding selenium. Monkey liver metallothionein was immunologically identical with human metallothionein. All the characteristics of MT_c obtained in the present study reveal a similarity between monkey and human metallothionein probably due to closer phylogenetic relationship between the two species.