大肠杆菌抗铜启动子的亚克隆

大肠杆菌的抗铜质粒中含有两个抗铜启动子,PpcoA和PpcoE,它们均含有copperbox。为了证实copper box在抗铜作用中的重要性以及研究抗铜启动子的特性,以质粒pUCD615为报告载体进行了这两个启动子不同长度片段的亚克隆。限制性酶切和DNA序列分析的结果表明,这些片段的亚克隆都是成功的;此外报告基因lux-荧光酶活性测定的结果表明,两个不含有copper box的Ppco short-lux亚克隆均不表达荧光酶活性,而其他启动子片段的亚克隆则都具有较高的酶活性,说明这两个亚克隆不具有启动子功能,初步证明在抗铜启动子中copper box是不可少的。     

Is there a role for copper in neurodegenerative diseases?

Copper is an essential metal in living organisms; thus, the maintenance of adequate copper levels is of vital importance and is highly regulated. Dysfunction of copper metabolism leading to its excess or deficiency results in severe ailments. Two examples of illnesses related to alterations in copper metabolism are Menkes and Wilson diseases. Several proteins are involved in the maintenance of copper homeostasis, including copper transporters and metal chaperones. In the last several years, the beta-amyloid-precursor protein (beta-APP) and the prion protein (PrP(C)), which are related to the neurodegenerative disorders Alzheimer and prion diseases respectively, have been associated with copper metabolism. Both proteins bind copper through copper-binding domains that also have been shown to reduce copper in vitro. Moreover, this ability to reduce copper is associated with a neuroprotective effect exerted by the copper-binding domain of both proteins against copper in vivo. In addition to a functional link between copper and beta-APP or PrP(C), evidence suggests that copper has a role in Alzheimer and prion diseases. Here, we review the evidence that supports both, the role of beta-APP and PrP(C), in copper metabolism and the putative role of copper in neurodegenerative diseases.     

Copper uptake by two sympatric species of Killifish Fundulus heteroclitus (L.) and F. majalis (Walbaum)

Differential copper uptake within brain, visceral, gill, somatic and residual tissue was examined in the two sympatric species of killifishes Fundulus heteroclitus and F. majalis . Various salinities and copper concentrations were tested on both juvenile and adult stages to examine their effects on copper uptake. Salinity played a significant role in copper uptake in both species of killifishes. However, the effect of salinity was specific for each species and life history stage. Copper content of various tissues was directly related to the uptake pattern exhibited by whole adult fishes. Fundulus majalis brain tissue and F. heteroclitus muscle tissue were exceptions. Severe body lesions and subcutaneous haemorrhaging were observed after 2–3 day exposure to both 2 and 8 ppm copper.     

Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase

Wilson's disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. However, the mechanism of biliary copper excretion has not been fully clarified. We examined the effect of copper on the intracellular localization of the Wilson disease gene product (ATP7B) and green fluorescent protein (GFP)-tagged ATP7B in a human hepatoma cell line (Huh7). The intracellular organelles were visualized by fluorescence microscopy. GFP-ATP7B colocalized with late endosome markers, but not with endoplasmic reticulum, Golgi, or lysosome markers in both the steady and copper-loaded states. ATP7B mainly localized at the perinuclear regions in both states. These results suggest that the main localization of ATP7B is in the late endosomes in both the steady and copper-loaded states. ATP7B seems to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes.     

Atomic force microscopy examination of the topography of a hydrated bacterial biofilm on a copper surface

A bacterium, designated CCI#8, that was isolated from a corroded copper coupon colonized both polished and unpolished copper surfaces under batch culture conditions. Atomic Force Microscopy (AFM) images revealed that the biofilm was heterogeneous in nature, both in depth and in cell distribution. Bacterial cells were shown to be associated with pits on the surface of the unpolished copper coupons. These observations support previous studies that CCI#8 is associated with the pitting corrosion of copper.     

A quick microwave histochemical stain for copper

A rapid microwave method is described for staining copper in liver. This procedure was compared with a conventional method for copper. To this end, liver sections obtained from patients affected by several liver diseases associated with copper overload, were stained both with the standard rubeanic acid method for copper and with our modification of the same method, incorporating microwave treatment. Liver sections from a normal human newborn were used as a positive control. In Wilson's disease in the cirrhotic stage, copper was detected by the conventional method solely in periportal cells; following the microwave treatment, we were able to demonstrate copper in the whole lobule. In alcoholic cirrhosis, rubeanic acid stained copper only in a few periportal cells, while, by our modified method, copper was detected in almost all periportal hepatocytes. In chronic biliary tract disease, and in the newborn liver, copper was demonstrated in a few periportal cells by both the two histochemical procedures. In conclusion, although copper was detected by both procedures, a different degree of positivity was sometimes observed by using microwaves. Moreover, the microwave-treated sections showed more contrast and less artifacts. From a practical point of view, for the simplicity of employment and, above all, for its quickness (10 min), we suggest the use of our method in all conditions where copper overload is suspected.     

The effect of copper supplementation on the concentration of copper in the brain of the brindled mouse.

The brindled mutant mouse is a useful model to study Menkes kinky-hair syndrome. The metabolic dysfunctions in both human and rodent are related to insufficient levels of bioavailable copper. Recently, copper supplementation therapy has been able both to prevent the appearance of various neuropathological changes and to prolong the life of these mutant mice. The optimum conditions for supplementation have been shown to be two intraperitoneal injections on postnatal days 7 and 10. The present study reports on the brain copper concentrations before, during and after the intraperitoneal copper therapy. The results demonstrate that postnatal days 7 and 10 correspond to two important epochs in copper homoeostasis. The supplementation therapy seems to provide sufficient bioavailable copper to respond to the needs of the animal at these crucial time points. The results are discussed in terms of their importance to the human copper disorder.     

Developmental patterns of copper and zinc concentrations in mouse liver and brain: evidence that the gene crinkled (cr) is associated with an abnormality in copper metabolism

An abnormality in copper metabolism during both the prenatal and postnatal (preweaning) periods was found to be associated with the autosomal recessive gene ”crinkled“ (cr) in mice. Liver copper concentration was significantly lower in crinkled mice (cr/cr) than in littermate controls (+/?) from 18 days of gestation to 20 days after birth. Crinkled mice older than 20 days of age had liver copper concentrations similar to those of littermate controls. Liver zinc and brain copper and zinc were similar in crinkled and noncrinkled mice at all times tested. In both crinkled and noncrinkled mice, brain copper concentration increased during the suckling period, and liver copper concentration decreased.     

Potential Phytoextraction and Phytostabilization of Perennial Peanut on Copper-Contaminated Vineyard Soils and Copper Mining Waste

This study sought to evaluate the potential of perennial peanut (Arachis pintoi) for copper phytoremediation in vineyard soils (Inceptisol and Mollisol) contaminated with copper and copper mining waste. Our results showed high phytomass production of perennial peanut in both vineyard soils. Macronutrient uptakes were not negatively affected by perennial peanut cultivated in all contaminated soils. Plants cultivated in Mollisol showed high copper concentrations in the roots and shoots of 475 and 52 mg kg⁻¹, respectively. Perennial peanut plants showed low translocation factor values for Cu, although these plants showed high bioaccumulation factor (BCF) for both vineyard soils, Inceptisol and Mollisol, with BCF values of 3.83 and 3.24, respectively, being characterized as a copper hyperaccumulator plant in these soils. Copper phytoextraction from Inceptisol soil was the highest for both roots and entire plant biomass, with more than 800 mg kg⁻¹ of copper in whole plant. The highest potential copper phytoextraction by perennial peanut was in Inceptisol soil with copper removal of 2,500 g ha⁻¹. Also, perennial peanut showed high potential for copper phytoremoval in copper mining waste and Mollisol with 1,700 and 1,500 g of copper per hectare, respectively. In addition, perennial peanuts characterized high potential for phytoextraction and phytostabilization of copper in vineyard soils and copper mining waste.     

Essential role of copper in the activity and regular periodicity of a recombinant,tumor-associated,cell surface,growth-related and time-keeping hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ENOX2)

ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46 kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.     

The CopRS Two-Component System Is Responsible for Resistance to Copper in the Cyanobacterium Synechocystis sp. PCC 6803

Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the P(I)-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen.     

鱼体(去鳃)和鱼鳃对不同形态铜的积累特征

在实验室条件下研究实验鱼Ｐａｒａｃｈｅｉｒｏｄｏｎ对人工河水中不同形态的积累特征,对比了鱼体（去鳃）和鱼鳃对铜吸收量的差异,并探讨了鱼对铜的吸收机理。研究结果表明,实验鱼鳃部和体内铜积累量均随水相游离铜浓度增高,暴露时间增长而增加,但鳃部积累浓度较鱼体其余部分高一个数量级,其从水相富集铜的速率显著高于鱼体。     

Active Uptake of Copper and Zinc during Haemodialysis

The uptake of copper and zinc by patients undergoing regular haemodialysis has been assessed by measuring the dialysis fluid for copper and zinc concentration, and the blood entering and leaving the dialysis coil for red cell copper, plasma free copper, and plasma zinc levels during priming of the coil and subsequent haemodialysis, and by in-vitro studies.Red cells avidly removed copper from dialysis fluid when mixed with saline during priming, but did not take up copper during the haemodialysis. At both these stages plasma actively took up both copper and zinc from dialysis fluid, even against a concentration gradient, so that loss of these metals from plasma to dialysis fluid did not occur.In the dialysis systems investigated the sources of the copper in the dialysis fluid were the copper plumbing of the tap-water and the dialysis coil, and the major source of zinc was the zinc oxide of the adhesive plaster around the dialysis coil.     

Uptake and efflux of copper-64 in Menkes''-disease and normal continuous lymphoid cell lines.

The accumulation of copper over 2 h by normal lymphoid cells and those from Menkes'-disease patients (Menkes' cells) was found to be biphasic, with an initial phase of rapid uptake, an approach to steady state at around 40-60 min, followed by a further accumulation phase. The accumulation of copper was not diminished by the addition of a variety of metabolic inhibitors, suggesting that copper uptake is not an active process. The presence of carbonyl cyanide m-chlorophenylhydrazone in the culture medium stimulated the uptake and accumulation of copper in both normal and Menkes' cells to the same absolute level. This effect appeared to be specific for copper, since the accumulation of Zn and Cd was unaffected. Menkes' cells did not differ from normal in their initial rate of copper uptake. Analysis of the uptake curve suggested that the membrane transport of copper involves both passive and facilitated diffusion. Initial rate of efflux from the cells was approximated by two methods. Menkes' cells did not appear to be affected in this function. It seems likely that the basic defect in Menkes' disease involves a step in intracellular copper transport rather than the membrane transport of copper.     

Mobilization of copper among tissues in the estuarine crab Scylla serrata (Forskal) under imposed starvation

The quantitative changes in copper free and bound to proteins in haemolymph and different forms of copper in muscle and hepatopancreas under imposed starvation were studied in the estuarine mud crab Scylla serrata. During the course of starvation, both haemolymph copper free and bound to proteins significantly declined and the regression analyses of these data further revealed that the haemolymph copper-free proteins were more affected than copper-bound proteins. The multiple stress condition namely injury and exsanguination along with starvation resulted in an earlier release and/or degradation of both these proteins. Hepatopancreas periodically accumulates and releases copper during starvation. The copper levels in haemolymph and hepatopancreas during different days of starvation showed a close inverse relationship between these two tissues. These changes in hepatopancreas were predominantly reflected in the copper that exists in association with low molecular weight substances. It is found that the copper thus accumulated was partly released back into haemolymph and a fraction may be excreted. This study also indicates the major role played by the low molecular weight substances in accommodation, detoxification and mobilization of copper in the decapod hepatopancreas during imposed starvation.     

Biofilms and microbially influenced cuprosolvency in domestic copper plumbing systems

AIMS: To survey biofilm accumulation within domestic copper plumbing pipes in South Australian drinking water distribution systems and examine its role in copper solvation (cuprosolvency). METHODS AND RESULTS: Cold water copper pipes were sampled from two different plumbing systems receiving filtered and unfiltered potable water respectively. Biomass was quantified by total organic carbon measurements and viable cell counts and microbial activity by respirometry. Biofilm accumulation was related to water chemistry within the systems, particularly nutrients, alkalinity and conductivity, as well as water turbulence. Laboratory coupon experiments were used to determine the effect of extracted biofilm on copper solvation. Biofilms were shown to be capable of both increasing and decreasing aqueous copper concentrations in comparison to sterile controls. CONCLUSIONS: The results suggest that water quality may influence the accumulation of biofilms in copper plumbing systems, as well as potential cuprosolvency activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of biofilms in copper plumbing systems and their ability to influence aqueous copper concentrations has implications for both public health and the management of distribution systems.     

Copper tolerance in the green alga, Chlorella vulgaris

Abstract. The effect of sub-lethal concentrations of copper upon tolerant and non-tolerant strains of Chlorella vulgaris was investigated. Copper concentrations of 0.2 and 0.4 mg dm⁻³ increased the lag phase of both strains, the effect being greater in the non-tolerant strain. No difference was observed in the toxicity of copper to the photosynthetic rates of the isolated chloroplasts of either strain. However, significant differences were shown at the whole cell level.
Lower copper uptake was shown by the tolerant cells. In both strains initial uptake of copper was followed by a phase of desorption before cell division occurred. In cultures of both strains the concentration of ionic copper was decreased by complexation with extracellular organic material. Over a 14 day growth period more organic material was produced by the tolerant cells. The organic material produced by the tolerant cell formed organo-copper complexes which had a higher conditional stability constant.
It is proposed that the cell wall acts as a barrier to copper in the tolerant cells and prevents copper from affecting cell metabolism. Organo-copper complexation occurs at this barrier and this complex is then released into the medium.     

Marginal copper deficiency and atherosclerosis

Copper is an essential trace element in the maintenance of the cardiovascular system. Copper-deficient diets can elicit, in animals, structural and functional changes that are comparable to those observed in coronary heart disease. In this study, the effect of dietary-induced copper deficiency on aortic lesion development was measured by quantitative image analysis in C57BL/6 mice that are susceptible to diet-induced aortic lesions. The diets administered were severely copper deficient (0.2 mg/kg diet), marginally deficient (0.6 mg/kg diet), or copper adequate (6.0 mg/kg diet). Similarly, increased aortic lesion areas and elevated serum cholesterol were demonstrated in both deficient groups, compared with the copper-adequate group. Evidence for graded differences in copper status among the dietary groups was shown by the dose-response increase in liver copper concentration, copper-zinc superoxide dismutase and cytochrome-c oxidase activities, together with serum caeruloplasmin oxidase with increasing intakes of dietary copper. Despite the difference in copper status between the copper marginal and severely deficient groups, similar lesions found in both groups of mice suggest a threshold effect of copper deficiency on lesion formation.