斑点叉尾鲖对不同形式赖氨酸利用的比较研究

为比较斑点叉尾鲖(Ictalurus punctatus)对不同形式赖氨酸的利用效果, 设置了鱼粉含量5%、豆粕含量15%的正对照饲料, 及鱼粉含量2.5%、豆粕含量0的负对照饲料, 在负对照饲料基础上, 分别添加晶体赖氨酸盐酸盐、晶体赖氨酸硫酸盐以及微囊赖氨酸盐酸盐, 使其赖氨酸含量达到与正对照饲料一致的水平, 共配制5组等氮等能饲料, 饲养平均体重为(54.40.1) g的斑点叉尾鲖60d, 考察不同形式赖氨酸对斑点叉尾鲖生长、血清生化指标和蛋白质消化酶活性的影响。结果表明, 与负对照组相比, 添加晶体赖氨酸盐酸盐和晶体赖氨酸硫酸盐对斑点叉尾鲖的生长性能影响不显著(P0.05), 而添加微囊赖氨酸盐酸盐提高斑点叉尾鲖增重率20.7% (P0.05), 降低饲料系数16.0% (P0.05), 在增重率与饲料系数方面达到与正对照组基本一致的水平(P0.05)。与负对照组相比, 在饲料中添加晶体赖氨酸盐酸盐、晶体赖氨酸硫酸盐以及微囊赖氨酸盐酸盐对血清谷丙转氨酶、谷草转氨酶、碱性磷酸酶及肠蛋白酶活性的影响均不显著(P0.05), 但显著提高了胃蛋白酶活性(P0.05)。此外, 添加微囊赖氨酸盐酸盐还显著提高了肝胰脏蛋白酶活性(P0.05)。上述结果表明, 在低赖氨酸实用饲料中补充晶体赖氨酸盐酸盐或赖氨酸硫酸盐对斑点叉尾鲖的生长性能改善作用不显著(P0.05), 而补充微囊赖氨酸盐酸盐则能显著提高斑点叉尾鲖增重率, 降低饲料系数。     

斑点叉尾鲖对不同形式赖氨酸利用的比较研究

为比较斑点叉尾鲖(Ictalurus punctatus)对不同形式赖氨酸的利用效果,设置了鱼粉含量5%、豆粕含量15%的正对照饲料,及鱼粉含量2.5%、豆粕含量0的负对照饲料,在负对照饲料基础上,分别添加晶体赖氨酸盐酸盐、晶体赖氨酸硫酸盐以及微囊赖氨酸盐酸盐,使其赖氨酸含量达到与正对照饲料一致的水平,共配制5组等氮等能饲料,饲养平均体重为(54.4±0.1)g的斑点叉尾鲖60d,考察不同形式赖氨酸对斑点叉尾鲖生长、血清生化指标和蛋白质消化酶活性的影响。结果表明,与负对照组相比,添加晶体赖氨酸盐酸盐和晶体赖氨酸硫酸盐对斑点叉尾鲖的生长性能影响不显著(P0.05),而添加微囊赖氨酸盐酸盐提高斑点叉尾鲖增重率20.7%(P0.05),降低饲料系数16.0%(P0.05),在增重率与饲料系数方面达到与正对照组基本一致的水平(P0.05)。与负对照组相比,在饲料中添加晶体赖氨酸盐酸盐、晶体赖氨酸硫酸盐以及微囊赖氨酸盐酸盐对血清谷丙转氨酶、谷草转氨酶、碱性磷酸酶及肠蛋白酶活性的影响均不显著(P0.05),但显著提高了胃蛋白酶活性(P0.05)。此外,添加微囊赖氨酸盐酸盐还显著提高了肝胰脏蛋白酶活性(P0.05)。上述结果表明,在低赖氨酸实用饲料中补充晶体赖氨酸盐酸盐或赖氨酸硫酸盐对斑点叉尾鲖的生长性能改善作用不显著(P0.05),而补充微囊赖氨酸盐酸盐则能显著提高斑点叉尾鲖增重率,降低饲料系数。     

异育银鲫幼鱼对饲料中维生素K需求的研究

以不同维生素K水平(0.13、2.15、3.25、6.40、12、17.20和23.20 mg/kg饲料)的7种精制饲料喂养初始体重约为(2.17±0.01) g的异育银鲫(Carassius auratus gibelio)10周, 每个处理3个重复, 研究异育银鲫对维生素K的需求量。结果显示: 饲料中维生素K的添加可以明显降低摄食率, 饲料中维生素K含量为2.15 mg/kg时, 摄食率出现最大值, 之后显著下降(P<0.05), 在12 mg/kg时达到最低值。特定生长率随着维生素K的添加表现出升高的趋势, 饲料中维生素K含量为12 mg/kg时, 出现最大值, 但是差异不显著(P>0.05)。饲料中维生素K的含量从0.13 mg/kg升至3.25 mg/kg时, 饲料效率显著升高(P<0.05), 随着饲料中维生素K的进一步添加, 趋于稳定(P>0.05), 在12 mg/kg时达到最大值, 并且与特定生长率呈正相关关系(SGR=0.01 FE+0.95, R²=0.95)。血液红细胞数目随着饲料维生素K含量的增加先显著升高(P<0.05), 在6.40 mg/kg时达到最大值, 之后趋于稳定(P>0.05)。血红蛋白含量、血球容积比、血清钙含量与血液中红细胞数目表现出相似的趋势, 均在不添加维生素K组出现最低值, 但是差异不显著(P>0.05)。肝体比、肥满度及鱼体生化组成均不受饲料维生素K水平的影响(P>0.05)。分别对饲料效率、红细胞数目进行折线回归得出异育银鲫幼鱼对维生素K的最适需求量为3.73—6.72 mg/kg饲料。     

两种温度下大黄素对团头鲂生长、血液指标及肝脏 HSP70 mRNA表达的影响

实验采用2×2双因子设计, 研究两种温度下在饲料中添加大黄素对团头鲂(Megalobrama amblycephala)生长、血液生理反应及肝脏HSP70 mRNA表达的影响。选取300尾健康的团头鲂初均重(7.29±0.01) g, 两种温度下(25℃、30℃)分别投喂不添加和添加25 mg/kg的大黄素配合饲料, 养殖时间为77d。结果显示: 在长期30℃高温下, 鱼体增重率(WGR)和特定生长率(SGR)、红细胞积压(HCT)、乳酸脱氢酶(LDH)显著升高, 饵料系数(FCR)和脏体比(VSI)、谷草转氨酶(AST)、总胆汁酸(TBA)显著下降(P0.05); 添加25 mg/kg大黄素, SGR和肝体比(HSI)、红细胞积压(HCT)、乳酸脱氢酶(LDH)显著升高, 饵料系数(FCR)和摄食率(FR)显著下降(P0.05)。温度和大黄素对谷丙转氨酶(ALT)和血糖(GLU)无显著影响(P0.05), 对尿素(UREA)、甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、补体3 (C3)、补体4 (C4)和肝脏HSP70 mRNA的相对含量存在显著交互作用(P0.05)。与常温对照组相比, 高温对照组UREA、TG、TC显著下降; 在高温下, 添加大黄素组UREA、TG、TC有升高趋势(P0.05), C3、C4和肝脏HSP70 mRNA的相对含量显著升高(P0.05)。以上结果表明, 在饲料中添加25 mg/kg大黄素有助于缓解长期高温对团头鲂血液生理的影响, 提高补体水平和肝脏HSP70 mRNA的相对含量, 促进鱼体的生长。       

饲料蛋白质水平与投喂频率对大黄鱼生长、体组成及蛋白质代谢的影响

以初始体重为(13.640.18)g的大黄鱼( Pseudosciaena crocea R.) 幼鱼为实验对象, 采用32双因子实验, 研究饲料蛋白质水平(40%、45%、50%)和投喂频率(2次/d、1次/d)及其交互作用对其生长、体组成和蛋白质代谢的影响。养殖实验在海水浮式网箱中进行, 养殖周期为8周。结果表明: 饲料蛋白质水平和投喂频率对大黄鱼幼鱼的增重率(WGR)、特定生长率(SGR)和饲料转化率(FCR)均影响显著(P0.05)。在40%和45%蛋白质组, 1次/d投喂的大黄鱼幼鱼的WGR和SGR均显著低于2次/d投喂组, 而FCR则相反。在2次/d投喂时, 45%蛋白质组的大黄鱼幼鱼SGR显著高于40%蛋白质组, 但与50%蛋白质组差异不显著(P0.05)。而在1次/d投喂时, 50%蛋白质组的大黄鱼幼鱼SGR显著高于40%和45%蛋白质组。在两种投喂频率下, 随着饲料蛋白质水平提高, 鱼体水分含量均有升高趋势, 蛋白质含量显著升高而脂肪含量显著下降(P0.05)。饲料的蛋白质水平和投喂频率分别对大黄鱼幼鱼的肝脏指数(HSI)、内脏指数(VSI)和血清中的谷丙转氨酶(ALT)及谷草转氨酶(AST)均影响不显著(P0.05)。投喂频率对肝脏的ALT和AST的影响不显著(P0.05)。在同一投喂频率下, 肝脏ALT和AST均随着饲料蛋白质水平的增加而显著提高(P0.05)。饲料中的蛋白质水平和投喂频率对大黄鱼幼鱼的生长和FCR的影响存在显著的交互作用(P0.05), 而对血清和肝脏中的ALT和AST、HSI、VSI、肥满度(CF)以及体组成的影响均无交互作用。       

大黄鱼幼鱼对饲料硒的需求量

为确定大黄鱼(Larimichthys croceus)对饲料硒的需求量, 以Na2SeO3为饲料硒源, 配制6种饲料, 硒的添加水平分别为0(对照组)、0.05、0.2、0.4、0.6和0.9 mg/kg, 实测值分别为0.08、0.16、0.27、0.44、0.66和0.96 mg/kg。在海水浮式网箱中养殖初始体重为(9.140.09) g的大黄鱼幼鱼10周, 结果表明增重率(WG)、全鱼和骨骼中的硒含量随着饲料硒含量的升高而显著升高(P0.05)。当饲料硒含量分别超过0.27、0.66、0.66 mg/kg时, 这些指标的变化趋于平稳。饲料硒含量对存活率(SR)、饲料效率(FE)、体组成、肝体比(HSI)、脏体比(VSI)和肥满度(CF)都没有显著影响(P0.05)。在血清中谷胱甘肽过氧化物酶(GPX)活性、超氧化物歧化酶(SOD)活性和总抗氧化力(T-AOC)随着饲料硒含量的升高呈现先升高后稳定的趋势(P0.05), 并分别在饲料硒含量为0.44、0.44、0.16 mg/kg时达到最大值。肝脏中GPX活性、SOD活性、T-AOC、过氧化氢酶(CAT)活性和谷胱甘肽还原酶(GR)活性与血清中相应酶的活性有相同的趋势。在肝脏中谷胱甘肽硫转移酶(GST)活性随着饲料硒含量的升高呈现先降低后升高的趋势(P0.05), 并在饲料硒含量最高(0.96 mg/kg)时其活力取得最大值。以WG为评价指标, 得出大黄鱼幼鱼对饲料中硒的需求量为0.178 mg/kg。以全鱼和骨骼中硒含量、肝脏GPX活性为评价指标, 得出大黄鱼幼鱼对饲料中硒的最小需求量分别为0.575、0.387和0.440 mg/kg。       

饲料中糖/脂肪比例对瓦氏黄颡鱼生长、饲料利用、血糖水平和肝脏糖酵解酶活力的影响

以初始体重(4.20±0.02)g的瓦氏黄颡鱼(Pelteobagrus vachelli)幼鱼为实验对象,在流水系统中进行为期8周的摄食生长实验,探讨饲料中糖与脂肪比例(CHO∶L)对其生长、饲料利用、血糖水平和糖酵解酶活力的影响。饲料等氮(粗蛋白40%)等能(19 MJ/g),CHO∶L梯度为0.75—6.53。每种饲料随机投喂3桶鱼,每桶(50 L)放养40尾鱼。实验结果表明随着CHO∶L升高,瓦氏黄颡鱼特定生长率(SGR)先升高后降低,当CHO∶L为3.55时,SGR达最大,显著高于CHO∶L为0.75和6.53时的值(P<0.05)。饲料效率(FE)和蛋白效率(PER)在CHO∶L为1.30—3.55区间内都没有显著差异,但当CHO∶L为0.75或6.53时,FE和PER的值都显著降低(P<0.05)。脏体比(VSI)、肝体比(HSI)在各处理组间无显差异(P>0.05)。随CHO∶L的增加,全鱼粗蛋白含量先增加后降低,且在CHO:L为3.55时达到最大值,且显著高于CHO:L为0.75时的值(P<0.05)。而全鱼粗脂肪含量随CHO∶L增加而显著减少(P<0.05)。血糖和血浆总胆固醇水平在各处理组间无显著差异(P>0.05),而血浆甘油三酯水平随CHO∶L的增加而显著增加(P<0.05)。随CHO∶L的增加,瓦氏黄颡鱼肝脏葡萄糖激酶(GK)活力先增加后降低,且在CHO∶L为3.55时活力最大,显著高于CHO∶L为0.75时的值(P<0.05)。肝脏己糖激酶(HK)、丙酮酸激酶(PK)和磷酸果糖激酶(PFK-1)活力在各处理组间没有显著差异(P>0.05)。用二次多项回归模型拟合特定生长率和CHO∶L的关系,得到饲料最适CHO∶L为4.06。     

三种室内饲养鱼类肠道微生物群落PCR-DGGE指纹分析

以室内饲养的斑点叉尾(Ictalurus punctatus)、银鲫和异育银鲫(中科三号)(Carassius auratus gibelio)幼鱼为对象,通过PCR-DGGE指纹技术对其肠道微生物群落进行了探索研究。在三种鱼的肠道中检测到不同的PCR-DGGE指纹谱带,其中斑点叉尾的平均谱带数(7.5)相对于银鲫和异育银鲫的谱带数(分别为15和14)要少。基于PCR-DGGE指纹谱带及各谱带相对丰度的UPGMA聚类和MDS排序结果显示银鲫和异育银鲫肠道微生物相似性高,而与斑点叉尾的差异比较大;rank-abundance散点图及回归分析也显示斑点叉尾与银鲫、异育银鲫肠道微生物群落存在显著差异(P0.05),而银鲫和异育银鲫之间无显著差异(P=0.383)。在斑点叉尾肠道中检测到的菌群主要是变形杆菌,包括-变形杆菌和-变形杆菌;而银鲫和异育银鲫肠道中菌群主要包括梭杆菌属中的类群,还包括变形杆菌门中的气单胞菌属,以及一些未知的类群。以上结果均表明在所研究的三种鱼的幼鱼阶段,其肠道微生物组成在不同种类鱼中存在差异,且该差异受基因型的影响可能更大。       

饲料中添加溶菌酶对吉富罗非鱼生长、免疫-抗氧化功能及血清抗菌性能的影响

为研究在饲料中添加不同水平的溶菌酶制品对吉富罗非鱼(GIFT, Oreochromis niloticus)生长性能、免疫-抗氧化功能和血清抗菌性能的影响, 选用平均体重为(11.350.08)g的吉富罗非鱼960尾, 随机分为6组(每组4个重复, 每个重复40尾), 分别投喂基础饲料(对照组)和5种添加水平分别为18、36、54、72和90 mg/kg溶菌酶制品的试验饲料, 养殖周期为60d。结果表明: (1) 54 mg/kg溶菌酶添加组鱼的生长性能和饲料利用情况最优, 增重率和蛋白质效率均显著高于对照组, 饲料系数显著低于对照组(P0.05); 肝体比随溶菌酶添加水平的增加呈现下降趋势, 90 mg/kg添加组显著低于对照组(P0.05); 脾脏指数在36和54mg/kg添加组显著低于对照组(P0.05); 全鱼粗蛋白和粗灰分含量在54 mg/kg添加组均呈现较高水平, 显著高于对照组(P0.05)。(2)溶菌酶添加水平对罗非鱼的免疫-抗氧化能力产生影响, 54和72 mg/kg添加水平能显著提高鱼体血清和肝脏的超氧化物歧化酶、过氧化氢酶活性, 降低丙二醛含量(P0.05); 肝脏溶菌酶活性在54和72 mg/kg添加组均显著高于对照组(P0.05), 而血清溶菌酶活性随溶菌酶添加水平的增加呈现下降趋势(L90组除外), 显著低于对照组(P0.05)。(3)血清抗菌试验显示, 54和72 mg/kg溶菌酶添加组罗非鱼对大肠杆菌、金黄色葡萄球菌、嗜水气单胞菌和溶藻弧菌的抑制能力显著高于对照组(P0.05), 而对枯草芽孢杆菌的抵抗能力最低, 比对照组分别低34.71%和42.21% (P0.05)。结果表明, 在本试验条件下, 在吉富罗非鱼饲料中添加54 mg/kg溶菌酶制品可以改善其生长性能; 当添加水平为54和72 mg/kg时, 罗非鱼的免疫-抗氧化能力和血清抗菌性能均得到了显著提高。     

饲料中铜水平对凡纳滨对虾免疫相关基因表达和抗菌能力的影响

在饲料中添加0、30和50 mg Cu/kg饲料的蛋氨酸铜,投喂凡纳滨对虾(Litopenaeus vannamei)7、14和21d,检测对虾体组织铜蓄积、免疫相关基因(Toll受体mRNA和溶菌酶mRNA)表达水平和免疫抗菌能力的变化。结果表明:凡纳滨对虾肝胰腺铜含量随着饲料蛋氨酸铜添加量增加及投喂时间延长而显著增加(P0.05);对虾肌肉的铜含量显著低于肝胰腺的铜含量。饲料中铜水平对凡纳滨对虾肌肉、血淋巴及肝胰腺中溶菌酶活性无显著影响(P0.05)。对虾组织SOD活性因饲料中铜水平和投喂时间变化显著,添加30 mgCu/kg组对虾肌肉、血淋巴和肝胰腺中SOD活性在第21天时显著高于其他两组(P0.05)。饲料中铜水平对凡纳滨对虾鳃组织中溶菌酶mRNA表达水平无显著影响,但显著影响鳃组织Toll受体mRNA表达水平(P0.05)。第7天时凡纳滨对虾Toll受体mRNA表达水平随着饲料铜水平升高而显著升高(P0.05);第14和第21天时,Toll受体mRNA表达水平在摄食添加30 mg Cu/kg组最高。人工急性感染溶藻弧菌(Vibrioalginolyticus)实验表明,第7天时,摄食添加50 mg Cu/kg组凡纳滨对虾全致死时间和半致死时间长于未添加铜组和添加30 mgCu/kg组,但在第14天,摄食添加30 mg Cu/kg组的全致死时间和半致死时间最长。研究表明饲料铜添加水平不但影响组织中铜的蓄积,还影响凡纳滨对虾SOD活性和Toll受体mRNA表达水平,从而影响机体的抗弧菌能力。     

Pharmacokinetic Study of Copper (II) Acetylsalicylate

This study was aimed at determination of pharmacokinetic parameters of copper (II) acetylsalicylate (CAS). Ten volunteers received a 60-mg dose of CAS. Blood samples were collected just before and after 0.25, 0.5, 0.75, 1.0, 1.5, 2.5, 3.0, 3.5, 4.0, 4.5, 5.5, 7.0, 10, and 12.0 h of administration of the drug. The plasma samples were analyzed for CAS and its metabolites by a validated high-performance liquid chromatography method having a suitable lower limit of quantification. The dose of 60 mg was well tolerated without any adverse effect. The maximum plasma concentration of CAS was found to be 0.38 mg L(-1) with t (max) of 0.72 h. The plasma half-life, clearance, and volume of distribution of CAS were 8.67 h, 66.30 L h(-1) and 829 L kg(-1), respectively. The elimination of CAS, acetylsalicylic acid, copper salicylate, and salicylic acid follows the first order kinetics with r (2) 0.979, 0.880, 0.991, and 0.998, respectively. The study provided for the first time the pharmacokinetic data for CAS after oral administration of CAS. The data were found to be useful in understanding the claimed enhanced anti-inflammatory activity of the drug as compared with that of acetylsalicylic acid.     

Tryptophan Cu(I)–π interaction fine-tunes the metal binding properties of the bacterial metallochaperone CusF

The periplasmic metallochaperone CusF coordinates Cu(I) and Ag(I) through a unique site consisting of a Met₂His motif as well as a Cu(I)–π interaction between a nearby tryptophan, W44, and the metal ion. Through mutational analyses we investigate here the role that W44 in CusF plays in metal coordination. Nuclear magnetic resonance spectra show that the specificity of CusF for Cu(I) and Ag(I) is not altered by mutation of W44. X-ray absorption spectroscopy studies reveal that W44 protects the bound Cu(I) from oxidation as well as from adventitious ligands. Competition assays demonstrate that W44 does not significantly contribute to the affinity of CusF for metal, but that substitution of W44 by methionine, which forms a fourth Cu(I) ligand, substantially increases the affinity. These studies indicate that W44 is important in maintaining a moderate-affinity and solvent-shielded three-coordinate environment for Cu(I), which has implications for the function of CusF as a metallochaperone. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Orally active antioxidative copper(II) aspirinate: synthesis, structure characterization, superoxide scavenging activity, and in vitro and in vivo antioxidative evaluations

Ever since it was proposed that reactive oxygen species (ROS) are involved in the pathogeneses of various diseases, superoxide dismutase (SOD)-mimetic complexes have been intensively studied. We prepared copper(II) aspirinate [Cu₂(asp)₄] from Cu(II) and aspirin, which has been in use for many years as an antipyretic, an analgesic, and an anti-inflammatory agent. However, Cu₂(asp)₄ has been found to have additional activities, including anti-inflammatory, antiulcer, anti-ischemic/reperfusion agent, anticancer, antimutagenic, and antimicrobial activities. The activity of copper salicylate [Cu(sal)₂] was also compared with that of Cu₂(asp)₄. The structure of the Cu₂(asp)₄ was determined using X-ray structure analysis. Its SOD-mimetic activity was determined using cytochrome c, electron spin resonance (ESR) spectroscopy, and ESR spin trap methods. The activity of Cu₂(asp)₄ was slightly greater than CuSO₄ and copper acetate [Cu(ace)₂] and slightly less than that of Cu(sal)₂. The in vitro antioxidant activity, evaluated in human epithelial or transformed neoplastic keratinocyte cells, HaCaT, and normal dermal fibroblasts in terms of cell survival following ultraviolet B (UVB) irradiation, was significantly increased in the presence of Cu₂(asp)₄, Cu(sal)₂, and CuSO₄. Further, ROS generation following UVA irradiation in the skin of hairless mice following oral treatment with Cu₂(asp)₄ for three consecutive days was significantly suppressed compared to the vehicle- or Cu(ace)₂-treated mice. On the basis of these results, Cu₂(asp)₄ was observed to be a potent antioxidative compound possessing antioxidative activity in biological systems. In conclusion, Cu₂(asp)₄ is a potent antioxidative agent that may be useful for future treatment of diseases resulting from ROS.     

Mechanism of ATPase-mediated Cu+ Export and Delivery to Periplasmic Chaperones: THE INTERACTION OF ESCHERICHIA COLI CopA AND CusF*

Cellular copper homeostasis requires transmembrane transport and compartmental trafficking while maintaining the cell essentially free of uncomplexed Cu^2+/+. In bacteria, soluble cytoplasmic and periplasmic chaperones bind and deliver Cu⁺ to target transporters or metalloenzymes. Transmembrane Cu⁺-ATPases couple the hydrolysis of ATP to the efflux of cytoplasmic Cu⁺. Cytosolic Cu⁺ chaperones (CopZ) interact with a structural platform in Cu⁺-ATPases (CopA) and deliver copper into the ion permeation path. CusF is a periplasmic Cu⁺ chaperone that supplies Cu⁺ to the CusCBA system for efflux to the extracellular milieu. In this report, using Escherichia coli CopA and CusF, direct Cu⁺ transfer from the ATPase to the periplasmic chaperone was observed. This required the specific interaction of the Cu⁺-bound form of CopA with apo-CusF for subsequent metal transfer upon ATP hydrolysis. As expected, the reverse Cu⁺ transfer from CusF to CopA was not observed. Mutation of CopA extracellular loops or the electropositive surface of CusF led to a decrease in Cu⁺ transfer efficiency. On the other hand, mutation of Met and Glu residues proposed to be part of the metal exit site in the ATPase yielded enzymes with lower turnover rates, although Cu⁺ transfer was minimally affected. These results show how soluble chaperones obtain Cu⁺ from transmembrane transporters. Furthermore, by explaining the movement of Cu⁺ from the cytoplasmic pool to the extracellular milieu, these data support a mechanism by which cytoplasmic Cu⁺ can be precisely directed to periplasmic targets via specific transporter-chaperone interactions.     

Coordination chemistry of copper proteins: how nature handles a toxic cargo for essential function

Biological copper is coordinated predominantly by just three ligand types: the side chains of histidine, cysteine, and methionine, with of course some exceptions. The arrangement of these components, however, is fascinating. The diversity provided by just these three ligands provides choices of nitrogen vs. sulfur, neutral vs. charged, hydrophilic vs. hydrophobic, susceptibility to oxidation, and degree of pH-sensitivity. In this review we examine how the total number of ligands, their spatial arrangement and solvent accessibility, the various combinations of imidazole, thiolate, and thioether donors, all work together to provide binding sites that either enable copper to carry out a function, or safely transport it in a way that prevents toxic reactivity. We separate copper proteins into two broad classes, those that utilize the metal as a cofactor, or those that traffic the metal. Enzymes and proteins that utilize copper as a cofactor use high affinity sites of high coordination numbers of 4-5 that prevent loss of the metal during redox cycling. Copper trafficking proteins, on the other hand, promote metal transfer either by having low affinity binding sites with moderate coordination number ~ 4, or by having lower coordinate binding sites of 2-3 ligands that bind with high affinity. Both strategies retain the metal but allow transfer under appropriate conditions. Analysis of studies from our own lab on model peptides, combined with those from other labs, raises an interesting hypothesis that various methionine/histidine/cysteine combinations provide organisms with dynamic, multifunctional domains on copper trafficking proteins that facilitate copper transfer under different extracellular, subcellular, and tissue-specific scenarios of pH, redox environment, and presence of other copper carriers or target proteins.     

Copper resistance mechanisms in bacteria and fungi

Abstract: Copper is both an essential micronutrient and a toxic heavy metal for most living cells. The presence of high concentrations of cupric ions in the environment promotes the selection of microorganisms possessing genetic determinants for copper resistance. Several examples of chromosomal and plasmid copper-resistance systems in bacteria have been reported, and the mechanisms of resistance have started to be understood at the molecular level. Bacterial mechanisms of copper resistance are related to reduced copper transport, enhanced effiux of cupric ions, or copper complexation by cell components. Copper tolerance in fungi has also been ascribed to diverse mechanisms involving trapping of the metal by cell-wall components, altered uptake of copper, extracellular chelation or precipitation by secreted metabolites, and intracellular complexing by metallothioneins and phytochelatins; only the metallothionein chelation mechanism has been approached with molecular detail.     

Copper chaperones: function, structure and copper-binding properties

 Copper is an absolute requirement for living systems and the intracellular trafficking of this metal to copper-dependent proteins is fundamental to normal cellular metabolism. The copper chaperones perform the dual functions of trafficking and the prevention of cytoplasmic exposure to copper ions in transit. Only a small number of copper chaperones have been identified at this time but their conservation across plant, bacterial and animal species suggests that the majority of living systems utilise these proteins for copper routing. The available data suggest that each copper-dependent protein in the cell is served by a specific copper chaperone. Although copper chaperones cannot be substituted for one another in a given cell type, copper chaperones that deliver to the same protein in different cell types appear to be functionally equivalent. The majority of the copper chaperones identified thus far have an "open-faced β-sandwich" global fold with a conserved MXCXXC metal-binding motif. Specificity for a given copper-dependent protein appears to be mediated by the residues surrounding the copper-binding motif. Copper binds to such proteins as Cu(I) in a trigonal complex with three sulfur ligands. Only the copper chaperone specific for cytochrome-c-oxidase, Cox17, deviates from this design. Received: 12 October 1998 / Accepted: 7 December 1998     

Reverse regulation of hepatic ceruloplasmin production in rat model of myocardial ischemia

BackgroundCeruloplasmin (Cp) is a major copper-binding protein produced in the liver and delivers copper to extrahepatic organs. Patients with myocardial infarction are often featured by an elevation of serum copper concentrations due to copper efflux from ischemic hearts. The present study was undertaken to test the hypothesis that serum copper elevation leads to up-regulation of hepatic Cp in myocardial infarction.MethodsAdult male Sprague-Dawley rats were subjected to left anterior descending (LAD) coronary artery ligation to induce myocardial infarction. Serum copper and Cp levels, as well as changes in hepatic Cp and copper-transporting P-type ATPase (Atp7b), were determined from blood and liver samples collected on day 1, 4, or 7 after the operation.ResultsSerum copper concentrations were significantly increased on day 4 after LAD ligation, accompanied by an increase in serum Cp levels and activities. Concomitantly, the protein levels of Cp and copper exporter, Atp7b, were also significantly increased in the liver. Furthermore, inhibiting the increase of serum copper by a copper chelator, triethylenetetramine (TETA), effectively abolished the elevated Cp activity after LAD ligation.ConclusionThese results indicate that serum Cp elevation in response to myocardial ischemia most likely resulted from the increased hepatic Cp production, which in turn was more responsive to serum copper elevation than inflammatory response following myocardial ischemia.     

Characterizing cultivable soil microbial communities from copper fungicide-amended olive orchard and vineyard soils

Use of copper-based fungicides has led to an increase in the total Cu content in agriculture soils. The focus of this study was to determine fractionation of Cu and to investigate the structure and the diversity of cultivable bacterial communities in two vineyards (one 25 years old and one 2 years old), one olive orchard and two forest soils. All soils developed on an Oligocene sandstone. The concentration of total Cu in the old vineyard (176.6 mg kg⁻¹) and olive orchard (145.5–296.7 mg kg⁻¹) was from 5 to 10 times higher than in forest soils. The major amount of Cu was found bound to the humic substances in cultivated soils, whereas in forest soils Cu was found in the residual mineral fraction. A relationship was found between the number of cultivable Cu-tolerant bacteria and total Cu content in soil. In the cultivated soils, Cu had a toxicological effect on bacterial community, and thereby Cu-levels > to 145 mg kg⁻¹ could be a risk to soil biota. Microbial communities were analysed by community level physiological profiling (CLPP), using the Biolog system, and by the amplified ribosomal DNA restriction analysis (ARDRA) approach. Only when cell suspensions containing 10⁴ colony-forming units (c.f.u.) were inoculated in each well of Biolog EcoPlates it was possible to discriminate microbial communities from different soil samples. As expected, 16S ARDRA showed that cultivated soils had a lower microbial diversity in respect to forest soils.