Ontogenically regulated expression of metallothionein and its messenger RNA in chick liver

By electrophoretic and immunological assay the concentration of hepatic metallothionein in new born chick liver was found to be ontogenically modulated, reaching a peak accumulation per gram liver in fourth day of hatching and declining below the detection limit after second week postnatal. The protein was undetectable upto second week of incubation in egg-embryonic stage. The concentration of metallothionein mRNA shows drastic change during first few days after hatching. The greatest accumulation of metallothionein mRNA was detected in the one day new born chicks, which declined rapidly there after, and reduced to a barely detectable level. Metallothionein was also detected in the in vitro translated product of one day neonatal chick hepatic poly(A+) RNA by S-cysteine labelling and immunoprecipitation. The naturally occurring new-born chick liver metallothionein was found to be a zinc-metallothionein and the concentration of hepatic zinc in new-born chick was found to undergo drastic modulation during development, unlike some other chick tissues. Endogenous zinc ion mobilization can thus play a significant role in the developmental regulation of chick metallothionein expression.     

Dimerization specificity of adult and neonatal chicken skeletal muscle myosin heavy chain rods

The dimerization specificity of the recombinantly expressed and purified rod domain of adult and neonatal chicken myosin heavy chain was analyzed using metal chelation chromatography. Our results indicate that full-length adult and neonatal rods preferentially formed homodimers when renatured from an equimolar mixture of the two isoforms denatured in guanidine hydrochloride. The contribution made toward the dimerization specificity by subdomains of the rod has been addressed by making a chimeric protein consisting of the subfragment 2 (S2) region of the adult isoform and the light meromyosin region of the neonatal isoform. The proportion of heterodimers formed in exchange experiments between the chimera and the neonatal and adult rods rose with increase in the sequence homology between the two exchanging proteins. This suggests that multiple regions of the rod domain of chicken MyHC including S2 can contribute toward dimerization specificity.     

The processing of human proinsulin and chicken proalbumin by rat hepatic vesicles suggests a convertase specific for X-Y-Arg-Arg or Arg-X-Y-Arg sequences

Vesicles from rat and chicken livers contain very similar Ca2(+)-dependent proteases that respectively cleave (human) proalbumin at an Arg-Arg site and chicken proalbumin at an Arg-Phe-Ala-Arg site. Similar Ca2(+)-dependent proteases are also present in pancreatic secretory granules and cleave proinsulin at two sites, Arg-Arg and Lys-Arg. The mammalian liver processes a large variety of different proproteins and in order to assess its processing site requirements, we investigated the ability of rat hepatic vesicle extracts to cleave purified chicken proalbumin and human proinsulin. Despite having only a monobasic processing site, chicken proalbumin was cleaved faster than human proalbumin which not only contains a dibasic site, but has an identical propeptide to that of the rat's own proalbumin. Human proinsulin was processed by the rat liver extracts; however, no mature insulin was produced. Cleavage occurred in only one place, presumably the Arg-Arg site at the B-C chain junction. This suggests that the mammalian liver might not contain a Type II Lys-Arg-directed convertase, only a Type I Arg-Arg-specific enzyme. The Type I enzyme that cleaves human proalbumin appears to be the same activity that cleaves chicken proalbumin, suggesting a specificity for either X-Y-Arg-Arg or Arg-X-Y-Arg sequences. This proposal is in keeping with the processing site motif of some 16 different proproteins that are known to be processed in the liver and is entirely consistent with the known in vivo specificity of the enzyme defined by naturally occurring variants of human proproteins.     

Heavy metal adsorbents prepared from the modification of cellulose: a review

A number of industries currently produce varying concentrations of heavy metal laden waste streams with significant consequences for any receiving environmental compartment. In recent years, increasing emphasis has been placed on environmental impact minimisation and resulting from this the range and capability of natural and prepared materials capable of heavy metal removal has seen steady development. In particular considerable work has been carried out on the use of both natural materials and their modifications. These natural materials, in many instances are relatively cheap, abundant in supply and have significant potential for modification and ultimately enhancement of their adsorption capabilities. This review paper reviews the current state of research on the use of the naturally occurring material cellulose, its modified forms and their efficacy as adsorbents for the removal of heavy metals from waste streams. Adsorbents based on direct modification of cellulose are evaluated initially and subsequently modifications resulting from the grafting of selected monomers to the cellulose backbone with subsequent functionalisation are assessed. The heavy metal adsorption capacities for these modified cellulose materials were found to be significant and levels of uptake were comparable, in many instances, to both other naturally occurring adsorbent materials and commercial ion exchange type resins. Many of the modified cellulose adsorbents proved regenerable and re-usable over a number of adsorption/desorption cycles allowing recovery of the adsorbed heavy metal in a more concentrated form.     

Age-dependent variation for inducibility of metallothionein genes in mouse liver by cadmium

Cadmium is a toxic metal that induces the expression of metallothionein genes in many tissues and that binds avidly to metallothionein, a soluble transition metal binding protein. The present study examined the temporal pattern and magnitude of accumulation of metallothionein mRNA in liver of C57BL/6J mice of various ages treated with cadmium. In adult female mice, accumulation was dependent on the dosage level of cadmium and related to the concentration of this metal in liver. The accumulation of metallothionein mRNA in liver depended on age at exposure to cadmium. Intraperitoneal administration of 2 mg of cadmium per kg provoked small increases (two- to threefold) in levels of metallothionein mRNA in livers of 7- and 14-day-old mice. In contrast, cadmium treatment of 28- and 56-day-old mice resulted in 12- to 19-fold increases in levels of metallothionein mRNA in liver with maximum increases occurring 3 to 4 hr after treatment. Because similar patterns for the accumulation of cadmium of liver were found in 7-, 28-, and 56-day-old mice, observed age-dependent differences in induction of metallothionein mRNA in liver were probably not due to differences in the accumulation of cadmium in this organ. Taken together, these data suggest that tissue-specific factors controlling the expression of metallothionein genes may account for developmental variation in the inducibility of these genes by cadmium. Ontogenic variation in accumulation of metallothionein mRNA after cadmium treatment may be a factor in developmental variation in the acute lethality of cadmium in C57BL/6J mice.     

Myosin heavy chain expression during development and following denervation of fast fibers in the red strip of the chicken pectoralis

The myosin heavy chain composition of muscle fibers that comprise the red strip of the pectoralis major was determined at different stages of development and following adult denervation. Using a library of characterized monoclonal antibodies we found that slow fibers of the red strip do not react with antibodies to any of the fast myosin heavy chains of the superficial pectoralis. Immunocytochemical analysis of the fast fibers of the adult red strip revealed that they contain the embryonic fast myosin heavy chain rather than the adult pectoral isoform found throughout the adult white pectoralis. This was confirmed using immunoblot analysis of myosin heavy chain peptide maps. We show that during development of the red strip both neonatal and adult myosin heavy chains appear transiently, but then disappear during maturation. Furthermore, while the fibers of the superficial pectoralis reexpress the neonatal isoform as a result of denervation, the fibers of the red strip reexpress the adult isoform. Our data demonstrate a new developmental program of fast myosin heavy chain expression in the chicken and suggest that the heterogeneity of myosin heavy chain expression in adult fast fibers results from repression of specific isoforms by innervation.     

Metallothionein synthesis in foetal, neonatal and maternal rat liver

The synthesis of hepatic metallothionein relative to other cytosol proteins was measured by [35S]cysteine incorporation in foetal, neonatal and pregnant rats. The relative rate of hepatic metallothionein synthesis reached a maximum in foetal liver on days 18-21 of gestation. Metallothionein synthesis then declined until weaning, when adult levels were established. The rate of metallothionein synthesis was greater in pregnant rats at term than in nulliparous rats. To determine if circulating inducing agents could play a role in the regulation of metallothionein synthesis in foetal liver we treated pregnant rats with inducers at a time prior to the normal rise in foetal liver metallothionein synthesis. Injections of copper, cadmium or hydrocortisone to 17-day-pregnant dams failed to induce foetal metallothionein synthesis. In contrast, zinc injection to the dam was an effective inducer in the foetuses. Maternal laparotomy (performed to expose the foetus for direct injection of inducers) induced foetal metallothionein synthesis. Metallothionein synthesis in the livers of 17-day-gestation dams was induced by all metal injections and laparotomy but, surprisingly, not by hydrocortisone injection. Maternal adrenalectomy did not influence the subsequent normal elevation in foetal or maternal metallothionein synthesis. These results, in conjunction with previous reports, suggest that mobilization of zinc in serum during late gestation may regulate foetal and maternal changes in metallothionein synthesis.     

Assembly of avian skeletal muscle myosins: evidence that homodimers of the heavy chain subunit are the thermodynamically stable form

Using a double antibody sandwich ELISA we examined the heavy chain isoform composition of myosin molecules isolated from chicken pectoralis major muscle during different stages of development. At 2- and 40-d posthatch, when multiple myosin heavy chain isoforms are being synthesized, we detected no heterodimeric myosins, suggesting that myosins are homodimers of the heavy chain subunit. Chymotryptic rod fragments of embryonic, neonatal, and adult myosins were prepared and equimolar mixtures of embryonic and neonatal rods and neonatal and adult rods were denatured in 8 M guanidine. The guanidine denatured myosin heavy chain fragments were either dialyzed or diluted into renaturation buffer and reformed dimers which were electrophoretically indistinguishable from native rods. Analysis of these renatured rods using double antibody sandwich ELISA showed them to be predominantly homodimers of each of the isoforms. Although hybrids between the different heavy chain fragments were not detected, exchange was possible under these conditions since mixture of biotinylated neonatal rods and fluoresceinated neonatal rods formed a heterodimeric biotinylated-fluoresceinated species upon renaturation. Therefore, we propose that homodimers are the thermodynamically stable form of skeletal muscle myosin isoforms and that there is no need to invoke compartmentalization or other cellular regulatory processes to explain the lack of heavy chain heterodimers in vivo.     

Order of metal binding in metallothionein

Purified isoforms of rat liver apometallothionein were reconstituted in vitro with Cd and Zn ions to study the order of binding of the 7 metal sites in the two separate metal clusters, one containing four metal ions (cluster A) and the other containing three (cluster B). Reconstitution with 7 Cd ions resulted in a metalloprotein similar to induced Cd,Zn-metallothionein by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin, and the pH-dependent release of Cd. Proteolytic digestion of metallothionein reconstituted with suboptimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in the 4-metal center, cluster A. Upon saturation of the 4 sites in cluster A, binding occurs in the 3-metal center, cluster B. Samples reconstituted with 1 to 4 Cd ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain that is known to encompass the 4-metal center. Samples renatured with 5 to 7 Cd ions per metallothionein molecule showed decreasing quantities of alpha fragment and increasing amounts of native-like metallothionein. Similar results were obtained in reconstitution studies with Zn ions. Samples reconstituted with 7 Cd eq followed by incubation with EDTA revealed that cluster B Cd ions were removed initially. The binding process in each domain is cooperative. Reconstitution of apometallothionein with 2 Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4 alpha cluster. Likewise, when Cd5-renatured metallothionein was digested with subtilisin, 30% of the molecules were identified as Cd7 metallothionein with the remainder as Cd4 alpha fragment.     

Physicochemical ProPerties and binding-site amino acid residues of galactoside-binding Protein of human Placenta

The galactose-binding lectin of human Placenta has been Purified to homogeneity by affinity chromatograPhy on asialo-fetuin column. The Protein, extractable from the tissue only with lactose is aPParently membrane-bound. Molecular weight determination of native Protein and subunit indicated a dimer of l3.4 kDa subunits. Inhibition of haemagglutination with various saccharides indicate that thiodigalactoside is the best inhibitor followed by lactose. However,P-nitroPhenyl-and 1-O-methyl derivatives of galactose showed that α-anomers inhibited slightly better than β-anomer. Modification of amino acid residues indicated involvement of arginine, lysine and histidine residues at the saccharidebinding site. Cysteine residue modificatioin also abolished haemagglutinating activity. Amino acid comPosition of the lectin is also Presented.     

人肝金属硫蛋白-I_A基因在鱼腥藻中的克隆与表达

将人工合成的人肝金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ,简称ＭＴ）－ＩＡ基因插入至中间载体ｐＲＬ－４３９上强启动子ｐｓｂＡ后,再将其与穿梭载体ｐＫＴ－２１０相连,得到大肠杆菌－蓝藻穿梭表达载体ｐＫＴ－ＭＴ,用三亲接合转移法将ｐＫＴ－ＭＴ转入丝状体蓝藻－鱼腥藻７１２０,经链霉素筛选,得到了稳定的转人肝ＭＴ－ＩＡ基因鱼腥藻．纯化单藻落,液体扩大培养．从鱼腥藻中提取的质粒经Ｓｏｕｔｈｅｒｎ印迹分析,确定人肝ＭＴ－ＩＡ基因已转入鱼腥藻７１２０中,Ｗｅｓｔｅｒｎ印迹分析表明,金属硫蛋白在转人肝ＭＴ－ＩＡ基因鱼腥藻中得到了表达．经原子吸收光谱法测定表达量约为７００μｇＭＴ／ｇ鲜藻,重金属耐受性实验表明,得到了能耐受重金属－镉的转人肝ＭＴ－ＩＡ基因鱼腥藻,它将在清除水域中重金属污染和医药研究方面发挥重要作用．     

The interaction of polyphosphoinositols with AMP deaminase

Polyphosphoinositols coupled to epoxy-activated Sepharose retained chicken liver AMP deaminase in a similar manner as phosphocellulose. After elution from polyphosphoinositol-Sepharose, in contrast to inositol-Sepharose and phosphocellulose, low Km AMP deaminase from the chicken liver exhibited markedly elevated S0.5 value. Several commercially available polyphosphoinositols were tested with rat liver AMP deaminase and only 1,3,4,5 IP4 was found to stimulate the enzyme. This is the first report on the effect of naturally occurring polyphosphoinositol derivative on the soluble enzyme.     

Elevated concentrations of naturally occurring heavy metals inversely correlate with reproductive output and body mass of the Kagu Rhynochetos jubatus

To assess the effects of naturally occurring heavy metals on wild birds, we compared reproductive success and heavy metal contents in feathers of Kagu Rhynochetos jubatus living on ultramafic (rich in heavy metals) soil with those of Kagu living on non‐ultramafic soil. From 2003 to 2016, we monitored breeding of 19 Kagu families by radiotracking and video‐monitoring, and collected rump down feathers from 69 wild Kagu. The metal concentrations in Kagu feathers correlated with the concentrations in the soil. The mean numbers of eggs laid and fledglings per year of Kagu families on non‐ultramafic soil were about four times higher, and home‐ranges three times smaller, than those of Kagu on ultramafic soil. Mass of eggs and the proportion of eggs that developed to fledglings were similar in the two areas, whereas the mass of adult Kagu on non‐ultramafic soil was nearly 10% higher than that of adult Kagu living on ultramafic soil. The impact of naturally occurring heavy metals on Kagu breeding productivity and body mass appears to act through their effects on food supply rather than being caused directly by metal toxicity. The results imply that conservation of Kagu might be more effective in non‐ultramafic areas, as populations can recover much faster on these soils and Kagu can then recolonize and bolster populations in ultramafic areas.     

Amino acids and peptides. X. Synthesis of two dotriacontapeptides corresponding to C-terminal sequence 30–61 (fragment α) of human liver metallothionein II and their heavy metal binding properties

Two kinds of dotriacontapeptides corresponding to C-terminal sequence 30–61 of human liver metallothionein, both containing 11 cysteine residues, were synthesized by the conventional fragment condensation method employing the HF deprotection or MSA deprotection method at the final step. Their heavy metal (Zn, Cd, or Cu) binding activity was examined, and it was found that their heavy metal binding properties were quite similar to those of native human liver thionein.     

Metallothionein mRNA expression in fetal mouse organs

The regulation of metallothionein biosynthesis in mammalian development was investigated by examining organs of 17-day fetal mice for biologically active metallothionein mRNA. Metallothionein was identified in cell-free translation products by migration in polyacrylamide gels and its characteristic elution on Sephadex G-50 columns. Metallothionein constitutes ～7.5% of [³⁵S]cysteine incorporated into polypeptides directed by mRNA from fetal liver, but it is not detectable in mRNA-directed products of fetal kidney, small bowel, heart, or adult liver. Consistent with a fetal-specific role, hepatic metallothionein mRNA content decreases abruptly in newborn mice, becoming undetectable within 12 days.     

Identification of metaflothionein in Pleurodeles waltl

The characterization of metallothionein in the Urodele amphibian species Pleurodeles waltl was achieved. A simple and rapid method for identification of metallothionein, based on its strong affinity for cadmium (109Cd), was used. We were able to show that metallothionein is constitutively synthesized in liver, ovary and brain. The property of metallothionein to strongly bind essential (Zn, Cu) as well as toxic (Cd, Hg) metals is consistent with a dual role in cellular metabolism, ie. homeostatis and detoxification of heavy metal ions.     

Levels of metallothionein messenger RNA in foetal, neonatal and maternal rat liver

We have used translation in vitro of hepatic polyadenylated RNA to characterize the levels of metallothionein mRNA in foetal, neonatal, pregnant and nulliparous rats. The translation products of foetal hepatic metallothionein mRNA increased relative to other mRNA translation products from day 18 of gestation to birth and attained a maximum, maintained throughout suckling, which is tenfold above 17-day foetal hepatic levels and fourfold above adult levels. Maternal liver metallothionein mRNA decreased fivefold between 17 days and 20 days of gestation, rose sharply immediately before birth, and was low throughout lactation.     

Metal ion binding properties of <Emphasis Type="Italic">Tricium aestivum</Emphasis> E<Subscript>c</Subscript>-1 metallothionein: evidence supporting two separate metal thiolate clusters

Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties and metal thiolate cluster formation of the Tricium aestivum (common wheat) early cysteine-labeled plant metallothionein (E_c-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant E_c-1 is able to bind a total of six divalent d ¹⁰ metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily so far. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This article is dedicated to Prof. Bernhard Lippert on the occasion of his 60th birthday.