A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein

The cellular form of the prion protein (PrP^C) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrP^C proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109↓His-110 (mouse sequence), termed α-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. β-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the α-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu²⁺ redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu²⁺ redox-generated reactive oxygen species do produce fragmentation corresponding to β-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu²⁺- and Zn²⁺-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of α-cleavage, Lys-109↓His-110 (MoPrP sequencing); however, upon addition of Cu²⁺, the location of α-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in α-cleavage at Lys-109↓His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119↓Val-120, with additional cleavage by ADAM10 at Gly-227↓Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrP^C fragmentation responds to Cu²⁺ and Zn²⁺.     

Damage to the cytoplasmic membrane of Escherichia Coli by catechin-copper (II) complexes

In the presence of a nonlethal concentration of Cu(II), washed Escherichia coli ATCC11775 cells were killed by (-)-epigallocatechin (EGC) and (-)-epicatechin (EC). Cell killing was accompanied by a depletion in both the ATP and potassium pools of the cells, but the DNA double strand was not broken, indicating that the bactericidal activity of catechins in the presence of Cu(II) results from damage to the cytoplasmic membrane. Induction of endogenous catalase in E. coli cells increased their resistance to being killed by the combination of catechins and Cu(II). In all cases studied, EGC and EC with Cu(II) were found to generate hydrogen peroxide, but its concentration was too low to account for the bactericidal activity. The bactericidal activity of EGC in the presence of Cu(II) was completely suppressed by ethylenediaminetetraacetate, bathocuproine, catalase, superoxide disumutase (SOD), heated catalase, and heated SOD, but not by dimethyl sulfoxide. When catalase, either heated or unheated, was added to the cells incubated with EGC in the presence of Cu(II), it completely inhibited further killing of the cells. These findings suggest that recycling redox reactions between Cu(II) and Cu(I), involving catechins and hydrogen peroxide on the cell surface, must be important in the mechanism of the killing.     

用于脑缺血小鼠海马SOD1表达的改进灌注固定法

常规灌注固定法多用于兔和大鼠等较大动物,并存在一些不足。改进了灌注固定法流程、灌注溶液的配方、流速、用量以及灌注装置,将其用于在显微操作下制备的缺血再灌注C57BL/6N小鼠模型,并对其海马进行H.E染色和免疫组织化学SOD1基因表达。结果显示,改进的灌注固定法使组织切片结构更加清晰,海马免疫阳性神经元定位于胞浆。缺血再灌注组(24hI/R)海马神经元SOD1表达比假手术对照组(sham-o)减少,而高压氧治疗组(24hHBO)SOD1表达有所恢复。表明改进的灌注固定法用于缺血再灌注C57BL/6N小鼠海马SOD1基因表达效果良好,结果可靠。实验结果提示,高压氧的治疗机制之一可能是通过增加SOD1基因表达而实现的。     

Characterization and catechole oxidase activity of a family of copper complexes coordinated by tripodal pyrazole-based ligands

A family of tripodal pyrazole-based ligands has been synthesized by a condensation reaction between 1-hydroxypyrazoles and aminoalcohols. The diversity was introduced both on the substituents of the pyrazole ring and on the side chain. The corresponding copper(II) complexes have been prepared by reaction with CuCl₂ in tetrahydrofuran. They have been characterized by EPR, UV spectroscopy and cyclic voltammetry. The absence of the half-field splitting signals in EPR suggests that the complex exists in solution as mononuclear species. The influence of substituents and side chain of the tripodal ligand on the catecholase activity of the complexes was studied. The reaction rate depends on two factors. First, the presence of an oxygen atom in the third position of the side chain should be avoided to keep the effectiveness of the reaction. Second, the electronic and steric effects of substituents on the pyrazole ring strongly affect the catalytic activity of the complex. Thus, best results were obtained with complexes containing unsubstituted pyrazole based-ligands. Kinetic investigations with the best catalyst based on the Michaelis–Menten model show that the catalytic activity of the mononuclear complex is close to that of some dicopper complexes described in literature.     

[Cu4(H2O)4(dmapox)2(btc)]n · 10nH2O: The first two-dimensional polymeric copper(II) complex with bridging μ-trans-oxamidate and μ4-1,2,4,5-benzentetracarboxylato ligands: Synthesis, crystal structure and DNA binding studies

A two-dimensional copper(II) polymer with formula of [Cu₄(H₂O)₄(dmapox)₂(btc)]_n · 10nH₂O, where dmapox is the dianion of N,N′-bis[3-(dimethylamino)propyl]oxamide and btc is the tetra-anion of 1,2,4,5-benzenetetracarboxylic acid, was synthesized and characterized by elemental analysis, conductivity measurement, IR and electronic spectral studies. The crystal structure of the complex has been determined by X-ray single-crystal diffraction. The structure consists of crystallized water molecules and neutral two-dimensional copper(II) coordination polymeric networks constructed both by the bis-tridentate μ-trans-dmapox and tetra-monodentate μ₄-btc bridging ligands. Each btc ligand links four trans-dmapox-bridged binuclear copper(II) building blocks [Cu₂(H₂O)₂(trans-dmapox)]²⁺ and each binuclear copper(II) building block attaches to two btc ligands forming an infinite 2D layer which consists of 4+4 grids with dimensions of 13.563(5) × 15.616(5) Å. The environment around the copper(II) atom can be described as a distorted square-pyramid and the Cu?Cu separations through μ-trans-dmapox and μ₄-btc bridging ligands are 5.225 Å (Cu1-Cu1ⁱ), 5.270 Å (Cu2-Cu2ⁱⁱ), 6.115 Å (Cu1-Cu2), 9.047 Å (Cu1-Cu2ⁱⁱⁱ) and 10.968 Å (Cu1-Cu1ⁱⁱⁱ), respectively. Abundant hydrogen bonds among the crystallized, the coordinated water molecules, and the uncoordinated carboxyl oxygen atoms cross-link the two-dimensional layers into an overall three-dimensional channel-like framework. The interaction of the copper(II) polymer with calf thymus DNA (CT-DNA) has been investigated by using absorption, emission spectral and electrochemical techniques. The results indicate that the copper(II) polymer interacts with DNA strongly (K_b = 4.8 × 10⁵ M⁻¹ and K_sv = 1.1 × 10⁴) and the interaction mode between the copper(II) polymer and DNA may be the groove binding. To the best of our knowledge, this is the first report about the crystal structure and DNA-binding studies of a two-dimensional copper(II) polymer bridged both by the trans-oxamidate and btc ligands.     

Binding of oxindole-Schiff base copper(II) complexes to DNA and its modulation by the ligand

Previous studies on copper(II) complexes with oxindole-Schiff base ligands have shown their potential antitumor activity towards different cells, inducing apoptosis through a preferential attack to DNA and/or mitochondria. Herein, we better characterize the interactions between some of these copper(II) complexes and DNA. Investigations on its binding ability to DNA were carried out by fluorescence measurements in competitive experiments with ethidium bromide, using plasmidial or calf-thymus DNA. These results indicated an efficient binding process similar to that observed with copper(II)-phenanthroline species, [Cu(o-phen)₂]²⁺, with binding constants in the range 3 to 9 × 10² M^− 1. DNA cleavage experiments in the presence and absence of distamycin, a recognized binder of DNA, indicated that this binding probably occurs at major or minor groove, leading to double-strand DNA cleavage, and being modulated by the imine ligand. Corroborating these data, discrete changes in EPR spectra of the studied complexes were observed in the presence of DNA, while more remarkable changes were observed in the presence of nucleotides (AMP, GMP, CMP or UMP). Additional evidence for preferential coordination of the copper centers to the bases guanine or cytosine was obtained from titrations of these complexes with each nucleotide, monitored by absorption spectral changes. Therefore, the obtained data point out to their action as groove binders to DNA bases, rather than as intercalators or covalent cross-linkers. Further investigations by SDS PAGE using ³²P-ATP or ³²P-oligonucleotides attested that no hydrolysis of phosphate linkage in DNA or RNA occurs, in the presence of such complexes, confirming their main oxidative mechanism of action.     

Copper(II) complexes based on a new chelating 4-(3,5-diphenyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)pyrimidine ligand: Synthesis and crystal structures. Lone pair-π, C-H···π, π-π and C-H···A (A = N, Cl) non-covalent interactions

A new bidentate chelating pyrazolylpyrimidine ligand bearing a strong electron-donating substituent, i.e. 4-(3,5-diphenyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)pyrimidine (L) (Scheme 1), has been synthesized and used to obtain the copper(II) complexes by reaction with CuCl₂. The molar ratio Cu:L = 1:2 leads to isolation of a complex having CuL₂Cl₂ empirical formula, while the molar ratio Cu:L = 1:1 gives a complex with CuLCl₂ empirical formula. The crystal structure of L as well as the structures of both complexes were studied by single crystal X-ray diffraction. The crystal structure of CuL₂Cl₂ compound is formed by trans-[CuL₂Cl₂] mononuclear molecules. Surprisingly, in contrast to the previous compound having molecular structure, the crystal structure of CuLCl₂ consists of mononuclear [CuL₂Cl]⁺ complex cations and dinuclear [Cu₂Cl₆]²⁻ anions. Thus, formula of CuLCl₂ complex can be represented as [CuL₂Cl]₂[Cu₂Cl₆]. In both complexes molecules of L adopt bidentate chelating coordination mode through N² atom of pyrazole and N³ atom of pyrimidine rings forming five-membered CuN₃C metallocycles. Owing to C-H···N interactions and π-π-stacking L molecules form 2D network. In the structure of trans-[CuL₂Cl₂] there exist double lone pair(N(piperidine))-π(pyrimidine) interactions and C-H···Cl contacts resulting in the formation of 1D chains. Layered 2D structure of [CuL₂Cl]₂[Cu₂Cl₆] results from C-H···Cl, C-H···π and double lone pair(Cl([CuL₂Cl]⁺ complex cation)-π(pyrimidine) interactions.     

铜尾矿污染区土壤酶活性研究

对浙江省哩浦铜尾矿污染区土壤酶活性进行了研究,结果表明,尾矿区及其周边土壤环境受到不同程度的Cu、Zn、Pb、Cd污染,从尾矿库中心到外围重金属污染程度逐渐减轻,而土壤酶活性则不断提高,其中以脱氢酶和脲酶活性增加最明显,回归分析表明,单一脱氢酶、脲酶、酸性磷酸酶以及蛋白酶活性与重金属复合元素含量之间存在显著线性关系,主成分分析结果显示,尾矿区土壤酶信息系统的第一、二主成分方差贡献率之和达98．24％,以第一、二主成分建立了两个土壤总体酶活性指标,并用此指标对各供试样本进行空间分类,其结果与以重金属含量为依据的划分结果基本吻合,可见,采用酶活性构筑的土壤信息系统的总体酶活性来表征矿区土壤的重金属污染状况是可行的。     

Cloning,characterization and chromosomal localization of the Sus scrofa SLC31A1 gene

Copper is an essential element necessary for normal function of numerous enzymes in all living organisms. Uptake of copper into the cell is thought to occur through the membrane protein, SLC31A1 (CTR1), which has been described in a variety of species including yeast, human and mouse. In this study, we present cloning, gene structure, chromosomal localization and expression pattern of the Sus scrofa SLC31A1 gene, which encodes a 189 amino acid protein. The (SSC) SLC31A1 gene is organized in four exons and spans an approximately 2.3 kb genomic region. We have localized the gene to chromosome 1q28-q2.13 using a somatic cell hybrid panel. This region shows conservation of synteny with human chromosome 9, where the human SLC31A1 (CTR1) gene has been localized. Expression studies suggest that SLC31A1 mRNA is transcribed in all tissues examined.     

Increased insulin-like growth factor-I gene expression precedes left ventricular cardiomyocyte hypertrophy in a rapidly-hypertrophying rat model system

Chronic pressure overload leads to an increase in the size, i.e. hypertrophy, of cardiomyocytes in the heart. However, the molecular mechanisms underlying this hypertrophy are not understood. Insulin-like growth factor-I (IGF-I) synthesized locally in the heart is known to be associated with the hypertrophic process. So far, however, cardiac IGF-I gene expression in the widely used rat model system has only been shown to be increased when the hypertrophy induced by pressure-overload was already established. Therefore, the question of whether IGF-I serves as an initiating or early-enhancing factor for the cardiac hypertrophy remains unanswered. Here, cardiac hypertension and hypertrophy were rapidly induced in the rat by complete constriction of the abdominal aorta between the origins of the renal arteries. Carotid arterial systolic blood pressure remained unchanged in sham rats but increased rapidly in the pressure-overloaded constricted rats with a sustained hypertension established by 3 days. Hypertrophy of left ventricular (LV) cardiomyocytes in constricted rats also occurred by 3 days. However, this hypertrophy was preceded by increases in LV IGF-I mRNA and protein which occurred within 1 day. These results support the hypothesis that cardiac-synthesized IGF-I is an initiating or early-enhancing factor for hypertrophy of LV cardiomyocytes.