A Highly Sensitive Chelator for Metal Staining, Bromopyridylazo-Diethylaminophenol

2-(5-Bromo-2-pyridylazo)-5-diethylaminophenol has been found to be capable of staining very small amounts of metals, particularly copper, iron, lead, and zinc, in tissue. The differentiation of a target metal from interfering metals is achieved by using masking agents, polyphosphates, and potassium cyanide.     

Removal of heavy metals from aqueous solution using Rhizopus delemar mycelia in free and polyurethane-bound form

This study assesses the ability of mycelia of Rhizopus delemar (both free and immobilized on polyurethane foam) to remove heavy metals from single-ion solutions as well as from a mixture of them. All experiments were conducted using 0.5-5 mm solutions of CuSO4 x 5H2O, CoCl2-6H2O and FeSO4 7H2O. Mycelia immobilized on polyurethane foam cells showed some times increase in uptake compared with that of free cells. Metal ions accumulation from a mixed solution was decreased slightly for cobalt and iron and considerable for copper ions. Heavy metal uptake was examined in the immobilized column experiments and more than 92% heavy metal removal (mg heavy metals removed/mg heavy metals added) from a mixed solution was achieved during the 5 cycles. During these experiments, the dry weight of the immobilized cells was decreased by only 2%. These results showed that immobilized mycelia of Rhizopus delemar can be used repeatedly for removal of heavy metals from aqueous solutions.     

Removal of Pb2+ and Ni2+ by bio-sludge in sequencing batch reactor (SBR) and granular activated carbon-SBR (GAC-SBR) systems

Living bio-sludge from domestic wastewater treatment plant was used as adsorbent of heavy metals (Pb(2+), Ni(2+)) and its adsorption capacity was about 10-30% reduced by autoclaving at 110 degrees C for 10 min. The living bio-sludge acclimatized in synthetic industrial estate wastewater (SIEWW) without heavy metals showed the highest Pb(2+) and Ni(2+) adsorption capacities at 840+/-20 and 720+/-10 mg/g bio-sludge, respectively. The adsorbed Pb(2+) and Ni(2+) were easily eluted (70-77%) from bio-sludge by washing with 0.1 mol/l HNO(3) solution. The heavy metals (Pb(2+), Ni(2+)) removal efficiency of both SBR and GAC-SBR systems were increased with the increase of hydraulic retention time (HRT), or the decrease of organic loading. The SBR system showed higher heavy metals removal efficiency than GAC-SBR system at the same organic loading or HRT. The Pb(2+), Ni(2+), BOD(5), COD and TKN removal efficiencies of GAC-SBR system were 88.6+/-0.9%, 94.6+/-0.1%, 91.3+/-1.0%, 81.9+/-1.0% and 62.9+/-0.5%, respectively with industrial estate wastewater (IEWW) with 410 mg/l glucose, 5 mg/l Pb(2+) and 5 mg/l Ni(2+) under organic loading of 1.25 kg BOD(5)/m(3) d (HRT of 3 days). The bio-sludge quality (sludge volume index: SVI) of the system was less than 80 ml/g. The excess sludge from both SBR and GAC-SBR systems with SIEWW under the organic loading of 1.25-2.50 kg BOD(5)/m(3) d contained Pb(2+) and Ni(2+) at concentrations of 240-250 mg Pb(2+)/g bio-sludge and 180-210 mg Ni(2+)/g bio-sludge, respectively.     

胁迫时间与非毒性离子对重金属抑制拟南芥种子发芽及幼苗生长的影响

测定了Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺单一重金属胁迫对拟南芥种子发芽和幼苗生长的影响.结果表明,重金属对幼苗生长的毒性大于对种子发芽的毒性,以抑制种子发芽的IC₅₀为指标,4种重金属的毒性顺序为Hg²⁺＞Cd²⁺＞Pb²⁺/Cu²⁺,以幼苗生长为指标,则毒性顺序为:Cu²⁺＞Hg²⁺＞Cd²⁺/Pb²⁺,并随着胁迫时间延长,种子萌发率下降.此外,不同重金属在不同发芽时段对种子的毒性也不尽相同,Cd²⁺的毒性在种子吸水后的0～12 h大于12～24 h,而Hg²⁺毒性在12～24 h大于0～12 h,其中,种皮对减轻重金属毒性起着十分重要的作用.通过非毒性离子(Ca²⁺、Mg²⁺、K⁺、Na⁺)与重金属离子(Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺)交互作用对拟南芥种子发芽及幼苗生长效应的研究发现, mmol·L^-1的Ca²⁺、Mg²⁺、K⁺、Na⁺可以增强Hg²⁺对种子发芽的毒性,但对Cd²⁺的毒性却没有影响.对于幼苗来说,Ca²⁺、Mg²⁺、K⁺、Na⁺可以显著增强Hg²⁺的毒性,Ca²⁺可以缓解Cd²⁺的毒性,但却增加Cu²⁺的毒性,K⁺可以缓解Pb²⁺对幼苗的毒害作用.最后,本文对重金属的毒害机理进行了探讨.     

Use of oligotrophic bacteria for the biological monitoring of heavy metals

Oligotrophic bacteria exhibited active growth even in nutritionally deficient medium made with nutrient broth that had been diluted with distilled water, 1 : 10 000. The oligotrophic bacteria, Sphingomonas paucimobilis KPS01 and Burkholderia cepacia KPC01 and KPC02 were found to be highly susceptible to heavy metals and to be potentially useful as sensors for the assessment of toxicity. The susceptibility of the bacteria to metals was measured by incubating the bacteria with metals of varying concentrations in the nutritionally deficient medium at 30 degrees C for 24 h. Bacteria were considered susceptible when the growth inhibition rate (EC50 was more than 50% of the control. The EC50 value of Ag+, Pb2+ and Cd2+ was 10(5)mmol l(-1) and Zn2+, Cr3+, Cr6+, Cu2+ and Hg2+ was 10(-4) mmol l(-1) in S. paucimobilis KPS01. Other strains also showed similar results. No difference in the EC50 was found using either the chloride or sulphate forms of these metals. The optimum incubation time was 24 h and a longer incubation time did not necessarily lead to more inhibition. The EC50 value rose in proportion to the concentration of nutrition in media. Environmental samples were tested and 14 out of 88 samples inhibited the growth of S. paucimobilis KPS01.     

Mechanism of adsorption of hard and soft metal ions to Saccharomyces cerevisiae and influence of hard and soft anions.

The applicability of the hard-and-soft principle of acids and bases in predicting metal adsorption characteristics in a biological context was investigated for metabolism-independent uptake of the metal ions Sr2+, Mn2+, Zn2+, Cu2+, Cd2+, and Tl+ by Saccharomyces cerevisiae. Metal adsorption increased with external metal concentration (5 to 50 microM), although some saturation of uptake of the harder ions examined, Sr2+, Mn2+, and Zn2+, was evident at the higher metal concentrations. Cation displacement experiments indicated that, with the exception of Tl+, relative covalent bonding (H+ displacement) of the metals was greater at low metal concentrations, while weaker electrostatic interactions (Mg2+ plus Ca2+ displacement) became increasingly important at higher concentrations. These results were correlated with curved Scatchard and reciprocal Langmuir plots of metal uptake data. Saturation of covalent binding sites was most marked for the hard metals, and consequently, although no relationship between metal hardness and ionic/covalent bonding ratios was evident at 10 microM metal, at 50 microM the ratio was generally higher for harder metals. Increasing inhibition of metal uptake at increasing external anion concentrations was partially attributed to the formation of metal-anion complexes. Inhibitory effects of the hard anion SO42(-) were most marked for uptake of the hard metals Sr2+ and Mn2+, whereas greater relative effects on adsorption of the softer cations Cu2+ and Cd2+ were correlated with complexation by the soft anion S2O32(-). Inhibition of uptake of the borderline metal Zn2+ by SO42(-) and that by S2O32(-) were approximately equal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heavy metal resistance patterns of Frankia strains

The sensitivity of 12 Frankia strains to heavy metals was determined by a growth inhibition assay. In general, all of the strains were sensitive to low concentrations (<0.5 mM) of Ag(1+), AsO(2)(1-), Cd(2+), SbO(2)(1-), and Ni(2+), but most of the strains were less sensitive to Pb(2+) (6 to 8 mM), CrO(4)(2-) (1.0 to 1.75 mM), AsO(4)(3-) (>50 mM), and SeO(2)(2-) (1.5 to 3.5 mM). While most strains were sensitive to 0.1 mM Cu(2+), four strains were resistant to elevated levels of Cu(2+) (2 to 5 mM and concentrations as high as 20 mM). The mechanism of SeO(2)(2-) resistance seems to involve reduction of the selenite oxyanion to insoluble elemental selenium, whereas Pb(2+) resistance and Cu(2+) resistance may involve sequestration or binding mechanisms. Indications of the resistance mechanisms for the other heavy metals were not as clear.     

Effect of metal microelements on biochemical indices of probiotic bacteria

The influence of metal-microelements (Zn, Cu, Ag, Au) in colloid and ionic form on the main biochemical parameters (ATP-ase activity, transmembrane potential and respiratory activity) of E. coli G35 N#1-413 and Ent. faecalis G35 N#4-410 probiont-strains has been studied with the goal to create complex metal-bearing probiotic preparations. Monotonous dose-dependent inhibitory influence of all mentioned metals in ionic form on the bacteria functional activity has been established. Metals' colloids had certainly stimulating influence on microorganisms' biochemical parameters in the same concentration limits with the following maximum positive effects: for Zn - 2x10(-7)-2x10(-4) mg/ml, for Cu - 8.4x10(-7)-8.4x10(-6) microg/ml, for Ag - 23x10(-7)-23x10(-4) microg/ml, for Au - 5x10(-6) microg/ml by metals. It has been shown, that cultural characteristics of probiont-bacteria in the presence of both ionic and colloid forms of studied metals remain stable. Certain stimulation of probiont-bacteria' main enzymes and life-support processes by colloidal forms of the studied metals-microelements in determined concentration and particle dimension limits is the evidence of a possibility of metals' colloids application in probiotic preparations' composition in order to increase the resistance and functional activity of probiont-microorganisms.     

Quantitative requirements for exponential growth of Alcaligenes eutrophus.

Quantitative nutrient requirements for unrestricted autotrophic growth of Alcaligenes eutrophus were determined. Minimum saturating concentrations of Mg2+, SO42-, PO43-, Fe3+, and Na2+ for an optical density increase of 2 were 10(-4) M 8 X10(-5) M, 5 X 10(-4) to 6 X 10(-4) M, 10(-5) M, and 10(-7) to 2 X 10(-7) M, respectively. Trace metal requirements for cobalt, chromium, and copper were also demonstrated, but minimum concentrations could not be determined because other reagents contributed a high background of these metals. Under certain conditions an apparent response to zinc was observed, although other experiments suggest the zinc salt contained another metal that was required for growth. Poly-beta-hydroxybutyrate biosynthesis was shown to be initiated by a magnesium or sulfate deficiency as well as by a nitrogen or phosphate deficiency.     

Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.     

Zinc and copper modulate differentially the P2X4 receptor

The rat ATP P2X4 receptor was expressed in Xenopus laevis oocytes to assess the effect of zinc and copper as possible regulators of purinergic mechanisms. ATP applied for 20 s evoked an inward cationic current with a median effective concentration (EC50) of 21.4+/-2.8 microM and a Hill coefficient (nH) of 1.5+/-0.1. Coapplication of ATP plus 10 microM zinc displaced leftward, in a parallel fashion, the ATP concentration-response curve, reducing the EC50 to 8.4+/-1.8 microM (p < 0.01) without altering the receptor nH. The zinc potentiation was fast in onset, easily reversible, and voltage-independent and did not require metal preexposure. The zinc EC50 was 2-5 microM, with a bell-shaped curve. At concentrations of 100-300 microM, zinc produced less potentiation, and at 1 mM, it inhibited 50% the ATP current. The effect of zinc was mimicked by cadmium. In contrast, copper inhibited the ATP-evoked currents in a time- and concentration-dependent fashion, reducing the maximal current (Imax) without altering the EC50. The copper-induced inhibition was slow in onset, slowly reversible, and voltage-independent. Whereas coapplication of 300 microM copper plus ATP reduced Imax to 36.2+/-5%, the coapplication of, or 60-s preexposure by, 10 microM copper reduced Imax to 79+/-9.2% (p < 0.05) and 39.6+/-8.7% (p < 0.01), respectively. The inhibition was noncompetitive in nature and mimicked by mercury. Cobalt, barium, and manganese did not modify significantly the ATP-evoked current, demonstrating metal specificity. The simultaneous 1-min preapplication of both metals revealed that the 10 microM zinc-induced potentiation was obliterated by 10 microM copper, whereas 30 microM copper not only reduced the potentiation, but inhibited the ATP response. Following coapplication of both metals for 20 s with ATP, at least 100 microM copper was required to counteract the 10 microM zinc-induced potentiation. The simultaneous preincubation with both metals provided evidence for a noncompetitive interaction. We hypothesize the existence of metal binding site(s), which are most likely localized in the extracellular domain of the P2X4 receptor structure. These sites are selective and accessible to extracellular metal applications and bind micromolar concentrations of metals. The present results are compatible with the working hypothesis that trace metals, such as copper and zinc, are physiological modulators of the P2X4 receptor. The modulation of brain purinergic transmission by physiologically and toxicologically relevant trace metal cations is highlighted.     

Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.     

Dependence of the inhibitory effect of metal cations on rat liver mitochondrial Ca2+ uptake on their physico-chemical properties

It has been found that Sr2+, La3+ Mn2+ (10-50 microM) inhibit Ca2+ transport into mitochondria in a competitive manner. Cd2+ ions show the mixed type inhibition of this transport. The inhibitory constants (Ki, microM) of the metals cations effect on Ca2+ transport increases in such a sequence: La3+ (2,11), Cd2+ (10,36), Mn2+ (49,29), Sr2+ (66,43). The metals cations inhibitory effect has an insignificant dependence on their ionic radii. But it is good correlated with the series of metals cations, based on the stability constants of their complexes with acetate (r = -0.96), aspartic (r = -0.91) and glutaminic acids and their hydratation enthalpy (r = -0.78). These data reveal that hydratation of metals cations and their interaction with carboxyles of Ca(2+)-uniporter plays an important role in the process of Ca2+ transport into mitochondrial matrix space and its inhibition by the metals cations. The mixed type inhibition of mitochondrial Ca2+ uptake by Cd2+ seems to be caused by the partial de-energization of mitochondria owing to Cd2+ interaction with SH-containing respiratory chain components and pore-forming ligands of mitochondrial membrane.     

营养杯对长喙田菁在铅锌尾矿上的生长、固氮和重金属积累的影响

设置移栽时营养杯的有无及其大小作试验,研究长喙田菁在乐昌铅锌矿强酸化尾矿上的生长、固氮和积累重金属情况。结果表明,强酸性(pH<3)是限制植物定植的主要因素,在pH=5-7情况下,长喙田菁能在该尾矿库中定植、生长和固氮,表现出良好的适应性。未带营养杯移栽的长喙田菁在尾矿上生长84d,其株高117cm、茎基部直径1.35cm、单株生物量(干物质)20.2g、单位面积生物量(干物质)2828kghm-2、氮素积累量40kghm-2;带营养杯移栽的上述各指标分别达到140-144cm、1.59-1.68cm、36.6-38.8g、5124-5432kghm-2和77-107kghm-2,均显著高于未带营养杯处理的。长喙田菁根部铅、锌、铜、镉含量均最高,其次为茎,叶中最低;长喙田菁的4种重金属积累量为锌(186-221mgkg-1)>铅(96-145mgkg-1)>铜(17-30mgkg-1)>镉(3-4mgkg-1)。带营养杯移栽能有效提高长喙田菁的产量和氮积累量,且明显降低其体内的重金属含量。试验证明长喙田菁是较理想的铅锌矿尾矿废弃地植被重建的先锋植物。     

Disturbances on delta aminolevulinate dehydratase (ALA-D) enzyme activity by Pb2+, Cd2+, Cu2+, Mg2+, Zn2+, Na+, K+ and Li+: analysis based on coordination geometry and acid-base Lewis capacity

ALA-D (EC 4.2.1.24) is the first cytosolic enzyme in the haem metabolic pathway. Some metals compete with its major cofactor Zn(2+), modifying both enzyme structure and function. Our purpose was to contribute to the understanding of the biochemical role of metals such as Pb(2+), Cd(2+), Cu(2+), Mg(2+), Zn(2+), Na(+), K(+) and Li(+) on ALA-D, using chicken embryos as experimental model. Mg(2+) and Zn(2+) showed enzyme activation in yolk sac membrane (YSM) (113% at 10(-5) M Mg(2+) and from 10(-4) M Zn(2+)), and slight inactivation in liver. Cd(2+) and Cu(2+) caused a non allosteric inhibition in both tissues (100% from 10(-4) M). Surprisingly Pb(2+) was not such a strong inhibitor. Interference of cations during the Schiff base formation in enzymatic catalysis process is explained considering their Lewis acid-base capacity, coordination geometry and electron configuration of valence. Interactions among monovalent cations and biochemical substances are governed chiefly by its electrostatic potential. 0.1 M K(+) and 0.4 M Na(+) produced 30% of enzymatic inhibition by the interference on interactions among the functional subunits. Li(+) activated the YSM enzyme (130% at 10(-5) M) due to a more specific interaction. This study may contribute to elucidate for the first time the possible structural differences between the YSM and liver enzymes from chicken embryo.     

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases

The effects of divalent metal cations on structural thermostability and the inactivation kinetics of homologous class II d-xylose isomerases (XI; EC 5.3.1.5) from mesophilic (Escherichia coli and Bacillus licheniformis), thermophilic (Thermoanaerobacterium thermosulfurigenes), and hyperthermophilic (Thermotoga neapolitana) bacteria were examined. Unlike the three less thermophilic XIs that were substantially structurally stabilized in the presence of Co2+ or Mn2+ (and Mg2+ to a lesser extent), the melting temperature [(Tm) approximately 100 degrees C] of T. neapolitana XI (TNXI) varied little in the presence or absence of a single type of metal. In the presence of any two of these metals, TNXI exhibited a second melting transition between 110 degrees C and 114 degrees C. TNXI kinetic inactivation, which was non-first order, could be modeled as a two-step sequential process. TNXI inactivation in the presence of 5 mm metal at 99-100 degrees C was slowest in the presence of Mn2+[half-life (t(1/2)) of 84 min], compared to Co2+ (t(1/2) of 14 min) and Mg2+ (t(1/2) of 2 min). While adding Co2+ to Mg2+ increased TNXI's t(1/2) at 99-100 degrees C from 2 to 7.5 min, TNXI showed no significant activity at temperatures above the first melting transition. The results reported here suggest that, unlike the other class II XIs examined, single metals are required for TNXI activity, but are not essential for its structural thermostability. The structural form corresponding to the second melting transition of TNXI in the presence of two metals is not known, but likely results from cooperative interactions between dissimilar metals in the two metal binding sites.     

镉、铜和锌胁迫下黑藻活性氧的产生及抗氧化酶活性的变化研究

研究了不同Cd、Cu、Zn处理浓度对黑藻体内活性氧()产生及对抗氧化酶(SOD、POD、CAT)活性的分子毒理学效应以探讨高等水生植物抗氧化酶对重金属胁迫的反应。结果表明,三种重金属都不同程度地加快了产生速率;Cu使SOD、POD、CAT活性下降;Cd也都减弱了SOD和POD活性,而CAT活性在0.5—5mg/L处理浓度时增加;Zn对SOD活性也为抑制作用,当浓度为0.5—5mg/L时POD和CAT活性都上升。关联度分析发现Cd、Cu和Zn胁迫下黑藻起主要保护作用的分别为SOD、POD和CAT,而SOD最易受到影响。Cd、Cu处理下的叶绿素含量也都呈下降趋势,而0.5—5mg/L的Zn浓度刺激了叶绿素合成。所有Zn处理、0.5mg/L的Cu处理和0.5—1mg/L的Cd处理的叶绿素a/b值都大于对照值。除了Cu使可溶性蛋白含量减少外,0.5—5mg/L的Zn和0.5—1mg/L的Cd都使其含量增加。综合起来,Cu的毒性最强,其次为Cd,Zn最弱。致死阈浓度分别为:Cu:0.5—1mg/L;Cd:1—2mg/L;Zn:5—6mg/L。SOD是评价重金属对沉水植物毒性效应的灵敏指标。黑藻对水环境Cu污染反应敏感。       

Ni2+高效结合肽的筛选与作用研究

利用噬菌体随机十二肽库和金属亲和层析对重金属Ni2 进行结合肽筛选。经4轮生物淘洗、噬菌体扩增和DNA测序,获得一组多肽序列。GenBank Blast分析未发现同源序列,Clustal W多重序列比对也未找到Ni2 金属结合肽结合基序,但可能含有多聚组氨酸(His)2-5。噬菌体单克隆金属离子螯合树脂的亲和力测定和反筛、抑菌解毒试验表明:展示有金属结合肽的噬菌体不仅对Ni2 具有高亲和力,而且对其它金属离子也有作用,Cu2 、Ni2 、Co2 、Zn2 等金属离子对金属结合肽的亲和力显著高于Cd2 和Cr2 ,展示金属结合肽的噬菌体对重金属Ni2 具有一定的耐受和解毒作用。显微形态学观察也显示金属结合肽与金属螯合树脂的作用。对于了解重金属与多肽的相互作用机理以及环境重金属修复等均具有重要意义和价值。