Cloning and expression analysis of the sheep ceruloplasmin cDNA.

The cDNA encoding sheep ceruloplasmin (sCP) was isolated from a sheep liver cDNA library. The cDNA contig was 3530 nucleotides in length and encoded a protein of 1048 amino acids. The deduced amino acid sequence showed a high degree of conservation (87%) when compared to the human ceruloplasmin (hCP) sequence. Northern blot analysis of sheep tissue revealed that the sheep ceruloplasmin gene (sCP) was expressed primarily in the liver, but low levels of mRNA were detected in the hypothalamus, spleen and uterus. No sCP mRNA was detected in the cortex, heart, intestine or kidney. Expression was not significantly affected by hepatic copper content. Northern blot analysis of sheep liver during development demonstrated little sCP expression during fetal life, but significant levels of mRNA were observed after birth. Significantly, the developmental expression pattern of sCP was closely correlated with that of the sheep Wilson disease gene (sATP7B), suggesting that the expression of the two genes may be coordinated to ensure that copper is supplied to apoceruloplasmin. Overall, the structure and expression of sCP appeared similar to other mammals, suggesting that abnormalities in CP were not responsible for the unusual sheep copper phenotype.     

Identification of the copper chaperone SAH in Ovis aries: expression analysis and in vitro interaction of SAH with ATP7B

A clone encoding the putative copper chaperone protein Sheep Atx1 Homologue (SAH) was isolated from a sheep liver cDNA library. The 466-bp cDNA encoded a predicted protein of 68 amino acids, with 44 and 81% amino acid identity to the yeast Atx1 and human Atox1 copper chaperone proteins, respectively. The characteristic MTCxxC and KTGK motifs were conserved in SAH. Northern blot analysis revealed an abundant 0.5-kb mRNA in all tissues examined. Elevated hepatic copper content did not affect the level of SAH mRNA in the liver. Analysis of SAH mRNA in the developing liver revealed low levels of expression in the foetal period, with a steady increase to adult levels occurring during development. In vitro two-hybrid analysis demonstrated SAH interacted with the amino terminal portion of the sheep Wilson's disease protein (ATP7B). The extent of this interaction was significantly reduced by the addition of the copper chelator bathocuproine disulfonic acid to the media. These results suggest SAH is a functional copper chaperone that is able to interact with ATP7B in a copper-dependent manner to facilitate copper transport into the secretory pathway.     

Adaptation of the Blood Antioxidant Defence Mechanisms of Sheep with a Genetic Lesion Resulting in Low Red Cell Glutathione Concentrations

Finnish Landrace sheep with a genetic lesion which results in restricted cysteine transport across the red cell membrane have total glutathione concentrations in their red blood cells that are approximately 40% of those in normal sheep of the same breed. However, dimethyldisulphide-challenged red blood cells from both phenotypes produce an ESR-spin adduct at similar rates. The resistance of the low glutathione phenotype red cells to oxidant challenge is reflected by increases in the activities of antioxidant enzymes. Sheep with a genotypic disorder in cysteine transport may be a suitable model for studying the genetic expression of antioxidant enzymes in response to oxidant loads.     

GDF9 as a candidate gene for prolificacy of Small Tail Han sheep

Growth differentiation factor 9 (GDF9) which controls the fecundity of Belclare, Cambridge, Santa Ines, Moghani, Ghezel and Thoka ewes was studied as a candidate gene for the prolificacy of Small Tail Han sheep. According to the sequence of ovine GDF9 gene, six pairs of primers were designed to detect single nucleotide polymorphisms of two exons of GDF9 gene in both high fecundity breed (Small Tail Han sheep) and low fecundity breed (Dorset sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Only the products amplified by primers 2-1 and 2-2 displayed polymorphisms. For primer 2-1, three genotypes (AA, AB and BB) were detected in both sheep breeds. Sequencing revealed one silent mutation (G477A) in exon 2 of GDF9 gene in the BB genotype in comparison with the AA, which was known as G3 mutation of GDF9 gene in Belclare and Cambridge ewes. The relationship of least squares means for litter size was AA?>?AB?>?BB in Small Tail Han sheep (P?>?0.05). For primer 2-2, two genotypes (CC and CD) were detected in both sheep breeds. Sequencing revealed one novel single nucleotide mutation (G729T) in exon 2 of GDF9 gene in the CD genotype in comparison with the CC, which resulted in an amino acid change (Gln243His). The ewes with mutation heterozygous genotype CD had 0.77 (P?相似文献   

Decreased expression of peroxiredoxin 6 in a mouse model of ethanol consumption

Alcoholic liver disease is multifactorial and oxidative stress is believed to play an intimate role in the initiation and progression of this pathology. The goals of this study were to investigate the effect of chronic ethanol treatment on inducing hepatic oxidative stress and peroxiredoxin 6 expression. After 9 weeks of treatment with an ethanol-containing diet, significant increases in serum ALT activity, liver to body weight ratio, liver triglycerides, CYP2E1 protein expression, and CYP2E1 activity were observed. Chronic ethanol feeding resulted in oxidative stress as evidenced by decreases in hepatic glutathione content and increased deposition of 4-hydroxynonenal and 4-oxononenal protein adducts. In addition, novel findings of decreased PRX6 protein and mRNA and increased levels of carbonylated PRX6 protein were observed in the ethanol-treated animals compared to the pair-fed controls. Lastly, NF-kappaB activity was found to be significantly increased in the ethanol-treated animals. Concurrent with the increase in NF-kappaB activity, decreases in both MEK1/2 and ERK1/2 phosphorylation were also observed in the ethanol-treated animals compared to the pair-fed controls. Together, these data demonstrate that chronic ethanol treatment results in oxidative stress, implicating NF-kappaB activation as an integral mechanism in the negative regulation of PRX6 gene expression in the mouse liver.     

Proteomic analyzes of copper metabolism in an in vitro model of Wilson disease using surface enhanced laser desorption/ionization-time of flight-mass spectrometry

In Wilson disease, mutations in the ATP7B-gene lead to hepatic accumulation of copper that becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and acute liver failure. Several proteins are probably involved in dealing with the excess copper and oxidative stress. As a first step towards biomarker discovery and analyzes of copper metabolism in Wilson disease patients we characterized copper-induced changes in protein expression in cell lysates and culture media from an in vitro copper-overload model using surface enhanced laser desorption/ionization (SELDI) proteomics technology. HepG2 cells were cultured for 48 h with a physiological (0.5 microM) or a pathological (100 microM) copper concentration. Samples were applied to weak cation exchange (WCX) proteinchip arrays and chips were analyzed by time of flight (TOF)-mass spectrometry. Copper-coated IMAC chips were used to detect copper-binding proteins in cell lysate of copper depleted cells using buffers with increasing imidazole concentrations. Data from the 2 to 50 kDa range indicate that high extra-cellular copper substantially altered both intra-cellular protein expression as well as the composition of the secretome. In the lysate 15 proteins were found up-regulated, while 6 proteins were down-regulated. In culture media 21 proteins were increased while 4 proteins were decreased in abundance. Copper-coated protein chips revealed the presence of 18 high-affinity copper-binding proteins. Further identification is necessary to determine the exact cellular roles of the discovered proteins.     

Cellularity of organs in mature rams of different breeds

The relative importance of cell number and cell size in determining the mass of 16 organs and tissues in mature rams of six different breeds was studied through estimation of organ deoxyribonucleic acid (DNA) content. The mean fleece-free empty body weight (FFEBW) ranged from 54.6 +/- 0.3 kg for Camden Park Merinos to 76.7 +/- 1.6 kg for Strong Wool Merinos. For all organs, mass increased with FFEBW, but the relationship was significant across all sheep for only eight organs (blood, kidney, liver, abomasum, vastus lateralis muscle, skin, perirenal fat and triceps muscle). There were significant differences between breeds in the mass of 11 organs. With four (heart, rumen reticulum, small intestine and testicular fat) this difference was independent of breed differences in FFEBW, whereas with another four (kidney, abomasum, vastus lateralis muscle and skin), it was closely related to FFEBW. Breed differences in the mass of the remaining three organs (blood, liver and perirenal fat) were partly related to FFEBW and partly breed specific. Blood mass increased with FFEBW across all animals, but, within a breed, it declined as FFEBW increased. The increase in the mass of perirenal fat with FFEBW was significantly greater within a breed than between breeds. Cell number increased significantly with the mass of all organs except blood and brain. There were between-breed differences in the number of cells in seven organs (liver, heart, rumen reticulum, abomasum, small intestine, vastus lateralis muscle and skin), which, except for heart, were attributable to between-breed differences in organ mass. With heart, the increase in cell number with organ mass within a breed was greater than across all breeds. Cell size was significantly related to organ mass only with vastus lateralis muscle, spleen, perirenal fat and liver. The relationship for vastus lateralis muscle and spleen was negative, indicating that cells were smaller in larger organs. There were differences between breeds in cell size for heart, vastus lateralis and triceps muscles. These differences for heart and triceps muscle were breed specific, whereas for vastus lateralis muscle it was attributed to breed differences in organ weight. There was a 30-fold range in mean cell size across organs, with adipose tissue having the largest cells, muscle tissue intermediate and visceral tissues the smallest. In general, organ mass is positively related to FFEBW. Cell number, not cell size, is largely responsible for differences in organ mass between mature sheep of different breeds.     

Copper decreases gene expression of TNF-<Emphasis Type="Italic">α</Emphasis>, IL-10, and of matrix metalloproteinases MMP-2 and MMP-9 in isolated perfused rat livers

Copper is known to induce oxidative stress in a number of models. It was shown that many pathophysiological events were associated with oxidative stress. Further, oxidative stress can increase gene expression of cytokines and of metalloproteinases. We previously found that copper toxic effects in isolated perfused rat livers were associated with significant oxidative stress (as assessed by lipid peroxidation, protein oxidation and oxidative DNA damage, particularly at concentration of 0.03 mM of Cu²⁺ in the perfusate). Here we investigated gene expression of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10); matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in frozen liver tissue samples by the real-time PCR assay. Compared to controls, copper at concentration of 0.01 mM did not affect gene expression of TNF-α, IL-10, MMP-2 and MMP-9, whereas copper at concentration of 0.03 mM significantly decreased gene expression of all the four TNF-α, IL-10, MMP-2 and MMP-9 by 69%, 81%, 43%, and 62%, respectively. These results suggest that copper-induced oxidative stress in the isolated rat liver can lead to the suppression of gene expression. Because TNF-α and metalloproteinases are involved also in liver regeneration, the suppression of these genes by copper may be one of the mechanisms by which acute intoxication of animals and humans with copper may impair regenerative capability of the liver.     

Comprehensive proteomics analysis of autophagy-deficient mouse liver

Autophagy is a bulk protein degradation system for the entire organelles and cytoplasmic proteins. Previously, we have shown the liver dysfunction by autophagy deficiency. To examine the pathological effect of autophagy deficiency, we examined protein composition and their levels in autophagy-deficient liver by the proteomic analysis. While impaired autophagy led to an increase in total protein mass, the protein composition was largely unchanged, consistent with non-selective proteins/organelles degradation of autophagy. However, a series of oxidative stress-inducible proteins, including glutathione S-transferase families, protein disulfide isomerase and glucose-regulated proteins were specifically increased in autophagy-deficient liver, probably due to enhanced gene expression, which is induced by accumulation of Nrf2 in the nuclei of mutant hepatocytes. Our results suggest that autophagy deficiency causes oxidative stress, and such stress might be the main cause of liver injury in autophagy-deficient liver.     

Prion protein reduces both oxidative and non-oxidative copper toxicity

The prion protein is a membrane tethered glycoprotein that binds copper. Conversion to an abnormal isoform is associated with neurodegenerative diseases known as prion diseases. Expression of the prion protein has been suggested to prevent cell death caused by oxidative stress. Using cell based models we investigated the potential of the prion protein to protect against copper toxicity. Although prion protein expression effectively protected neurones from copper toxicity, this protection was not necessarily associated with reduction in oxidative damage. We also showed that glycine and the prion protein could both protect neuronal cells from oxidative stress. Only the prion protein could protect these cells from the toxicity of copper. In contrast glycine increased copper toxicity without any apparent oxidative stress or lipid peroxidation. Mutational analysis showed that protection by the prion protein was dependent upon the copper binding octameric repeat region. Our findings demonstrate that copper toxicity can be independent of measured oxidative stress and that prion protein expression primarily protects against copper toxicity independently of the mechanism of cell death.     

Effects of Different Copper Sources and Levels on Plasma Superoxide Dismutase, Lipid Peroxidation, and Copper Status of Lambs

This study was performed to determine the effects of different copper (Cu) sources and levels on plasma superoxide dismutase (SOD), lipid peroxidation, and Cu status of lambs. Fifty Dorper × Mongolia wether lambs (approximately 3?month of age; average BW?=?23.8?±?0.6?kg) were divided into five equal groups each with ten animals according to their weight. Treatments consisted of (1) control (no supplemental Cu), (2) 10?mg Cu/kg DM from Cu-lysine, (3) 20?mg Cu/kg DM from Cu-lysine, (4) 10?mg Cu/kg DM from tribasic copper chloride (Cu(2)(OH)(3)Cl; TBCC), and (5) 20?mg Cu/kg DM from TBCC. The Cu concentration was 6.74?mg/kg DM in the basal diet. Plasma copper concentrations and ceruloplasmin activities were not affected on day?30 by Cu supplementation. Copper supplementation increased plasma and liver copper concentrations and ceruloplasmin activities on day?60. Muscle Cu concentrations were not affected by Cu supplementation. There were no differences in plasma, liver, and muscle Cu concentrations and ceruloplasmin activities between Cu-lysine and TBCC. Liver copper concentrations and plasma ceruloplasmin activities were increased in lambs supplemented with 20?mg Cu/kg DM than in those supplemented with 10?mg Cu/kg DM on day?60. However, copper levels had no effects on Cu concentrations in plasma and muscle. Malondialdehyde (MDA) concentrations were decreased in plasma and liver tissues, but not affected in muscle by Cu supplementation. Plasma SOD activities were increased by Cu supplementation. There were no differences in plasma, liver, and muscle MDA concentrations and plasma SOD activities between Cu sources and levels. These results indicated that Cu supplementation increased plasma SOD activity, lipid oxidative stability, and copper status of lambs, but did not influence lipid oxidative stability in sheep muscle. Cu-lysine and TBCC were of similar availability when offered to finishing sheep.     

绵羊肌肉H-FABP和PPARγ基因表达的发育性变化及其对肌内脂肪含量的影响

选取不同日龄的雄性哈萨克羊和新疆细毛羊,屠宰后取背最长肌,用索氏抽提法检测肌内脂肪（Intamuscular fat,IMF）含量,用荧光实时定量PCR法检测心脏脂肪酸结合蛋白（heart fatty acid-binding protein,H-FABP）和过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptorγ,PPARγ）基因表达的发育性变化极其对IMF含量的影响.结果表明：（1）随着日龄的增加,IMF含量在雄性哈萨克羊中持续上升,且在各生长期间的差异显著（P＜0.05）,而在新疆细毛羊的各生长期间无显著差异（P＞0.05）.雄性哈萨克羊的IMF含量在30～90日龄间极显著高于新疆细毛羊（P＜0.01）;（2）雄性哈萨克羊肌肉H-FABP基因的表达量在2日龄时最高,30日龄时降到最低,然后持续上升,各日龄间差异显著（P＜0.05）.新疆细毛羊中的表达量在2日龄时也最高,然后持续下降,到90日龄时降到了最低点,然后上升,各日龄间差异也显著（P＜0.05）;（3）雄性哈萨克羊肌肉PPARγ基因的表达量随着日龄的增加而呈下降趋势,各日龄间差异显著（P＜0.05）;新疆细毛羊中的表达量在2～60日龄期间持续上升且各日龄间差异显著（P＜0.05）,但在90日龄时降到最低点,然后上升;（4）在哈萨克羊中,H-FABP基因在30～90日龄间的表达量与IMF含量的相关系数为0.737（P＜0.01）,而PPARγ基因在2-90日龄间的表达量与之的相关系数为-0.835（P＜0.01）.     

Cloning, mapping and expression analysis of the sheep Wilson disease gene homologue

Copper homeostasis in mammals is maintained by the balance of dietary intake and copper excretion via the bile. Sheep have a variant copper phenotype and do not efficiently excrete copper by this mechanism, often resulting in excessive copper accumulation in the liver. The Wilson disease protein (ATP7B) is a copper transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile. To investigate the role of ATP7B in the sheep copper accumulation phenotype, the cDNA encoding the ovine homologue of ATP7B was isolated and sequenced and the gene was localised by fluorescence in situ hybridisation to chromosome 10. The 6.3 kb cDNA encoded a predicted protein of 1444 amino acids which included all of the functional domains characteristic of copper transporting P-type ATPases. ATP7B mRNA was expressed primarily in the liver with lower levels present in the intestine, hypothalamus and ovary. A splice variant of ATP7B mRNA, which was expressed in the liver and comprised approximately 10% of the total ATP7B mRNA pool, also was isolated. The results suggest that ATP7B is produced in the sheep and that the tendency to accumulate copper in the liver is not due to a gross alteration in the structure or expression of ATP7B.     

Relevance of Non-ceruloplasmin Copper to Oxidative Stress in Patients with Hepatocellular Carcinoma

Altered copper homeostasis and oxidative stress have been observed in patients with hepatocellular carcinoma. Non-ceruloplasmin copper, the free form, is a potent pro-oxidant than the protein bound copper. The aim of the present study was to evaluate which form of copper can be correlated with the oxidative stress in the circulation and in the malignant liver tissues of hepatocellular carcinoma patients. Hepatocellular carcinoma patients (grades II and III, n = 18) were enrolled in this study. Serum levels of total, free and bound copper, ceruloplasmin, iron, iron-binding capacity, lipid peroxidation products, and enzymatic and non-enzymatic antioxidants were quantified in serum and in malignant liver tissues and compared with those of normal samples (n = 20). A significant positive correlation between the serum non-ceruloplasmin copper and lipid peroxidation products and negative correlation with antioxidants were observed in hepatocellular carcinoma patients. In liver tissue, glutathione peroxidase, superoxide dismutase, and catalase activity were significantly decreased with concomitant elevation in oxidative stress markers. Our experiment revealed that the elevation in non-ceruloplasmin copper has high relevance with the oxidative stress than the bound copper.     

Unfolding of population structure in Baltic sheep breeds using microsatellite analysis

Studies of domestic animals are performed on breeds, but a breed does not necessarily equate to a genetically defined population. The division of sheep from three native and four modern Baltic sheep breeds was studied using 21 microsatellite loci and applying a Bayesian clustering method. A traditional breed-wise approach was compared to that relying on the pattern of molecular diversity. In this study, a breed was found to be inconsistent with a distinct genetic population for three reasons: (i) a lack of differentiation between modern Baltic breeds, since the majority of the studied sheep formed a single population; (ii) the presence of individuals of foreign ancestry within the breed; and (iii) an undefined local Saaremaa sheep was referred to as a breed, but was shown to consist of separate populations. In the breed-wise approach, only the clearly distinct Ruhnu sheep demonstrated low within-breed variation, although the newly identified Saaremaa populations also have low variability. Providing adequate management recommendations for the Saaremaa sheep is not possible without further studies, but the potential harmful effects of inbreeding in the Ruhnu sheep could be reduced through the use of two genetically related Saaremaa populations. In other breeds, excessive crossing appears to be a larger concern than inbreeding. Assigning individuals into populations based on the pattern of genetic diversity offers potentially unbiased means of elucidating the genetic population structure of species. Combining these genetic populations with phenotypic and aetiological data will enable formulation of the most informed recommendations for gene resource management.     

Identification of proteins involved in intracellular copper metabolism. Low levels of a approximately 48-kDa copper-binding protein in the brindled mouse model of Menkes disease

Little is known about copper metabolism at the cellular level. The brindled mouse is an animal model of Menkes disease which is an inborn error of copper metabolism. Control and brindled mice were used to identify copper-binding proteins with possible roles in normal copper metabolism that are affected by the defect in the brindled mice. When 64Cu-labeled hepatic or renal cytosols from control mice were applied to Mono Q or Superose columns, a approximately 48-kDa protein coeluted with the protein fractions which contained the radiolabeled copper. Large decreases in copper binding were detected in these fractions from the brindled mice. The same column fractions which showed decreased copper binding showed large decreases in the levels of the approximately 48-kDa protein. Decreased copper binding and approximately 48-kDa protein were not simply secondary to the abnormal hepatic and renal copper levels that are found in the brindled mice since although their liver copper levels are low, their kidney copper levels are high. Elevated levels of an approximately 80-kDa heat shock protein were also detected in the hepatic and renal cytosols from the brindled mice. Consistent with expression of the primary defect in both the liver and kidney, the levels of the approximately 48- and approximately 80-kDa proteins were affected similarly in both organs. Irrespective of how the low levels of the approximately 48-kDa protein may be related to the basic defect in the brindled mice, the data are consistent with an important role for the approximately 48-kDa protein in intracellular copper metabolism.     

Milk ceruloplasmin is a valuable source of nutrient copper ions for mammalian newborns

This research focuses on the role of milk ceruloplasmin (Cp), the main extracellular copper-containing protein of vertebrates, as a source of copper for newborns. In the first part of the study, Cp concentration and Cp-associated copper were measured in human skimmed milk at the 1st and the 5th days postpartum. It was shown that most of the copper was associated with Cp and that the decrease in copper concentration during lactation was related to the drop of Cp levels. The following in vivo experiments demonstrated that milk [(125)I]Cp per os administered to 6-day-old rats (embryonic-type copper metabolism) was transported into their bloodstream. The electrophoretic mobility and relative molecular weight of [(125)I]Cp transferred through the cellular barrier remained unaltered. However, 22-day-old rats (adult-type copper metabolism) digested the administered milk [(125)I]Cp completely. In the final part of the study, newborn rats were fed with baby formula for 8d. It was found that these rats switched their copper metabolism from embryonic type to adult type earlier than their littermates fed by dams. Activation of Cp gene expression in the liver, increased Cp and copper concentrations in the blood, and reduced copper content of the liver were observed in the rats fed with baby formula. In the brain, no copper concentration change was observed, but Cp and copper concentrations were dramatically increased in the cerebrospinal fluid. The role of milk Cp as a source of copper adapted to embryonic-type copper metabolism is discussed.     

Genetic diversity of protein markers in sheep population from Ukraine

Based on polymorphism of genes for antigen factors of six blood-group systems and four blood protein loci, genetic structure and the main variation parameters were studied in three sheep breeds and three sheep breed types constituting the basis of purebred sheep resources in Ukraine. Specific features of the distribution of genotypes and alleles of polymorphic loci were determined in each of the studied sheep groups depending on their origin and production type. The molecular-genetic markers used in the analysis of the genetic relationships between the sheep breeds and breed types were shown to objectively reflect their breeding history and evolution. Integrally, each of the studied gene pools had a specific profile of gene frequencies reflecting breeding specificity, breed history, and genetic differentiation of breeds.