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Copper deficiency in adult rats was induced by addition of silver chloride to the feed. The concentrations of silver, copper, iron, and zinc and relative activity of genes for copper transporting proteins and copper enzymes were measured in the cortex, cerebellum, hippocampus, amygdala, pituitary gland, and hypothalamus. Silver was accumulated only in the hypothalamic-pituitary system. These changes were accompanied by a decrease in the concentration of copper and increase in the contents of iron and zinc. Activity of genes for copper transport enzymes (high-affinity copper transporter; and two copper transport ATPases, ATP7A and ATP7B) and copper enzymes that were formed in the intracellular secretory pathway did not decrease in the brain of rats with copper deficiency. Relative activity of genes for intracellular copper enzymes (Cu(2+)/Zn(2+) superoxide dismutase and subunit IV of cytochrome c oxidase), concentration of immunoreactive polypeptides of superoxide dismutase, and enzymatic activity of superoxide dismutase remained unchanged under these conditions.

Fibrillogenesis was induced in rats by injection of a fragment of neurotoxic protein, beta-amyloid protein precursor, into the cerebral ventricle. Copper, iron, and zinc concentrations and relative activities of genes of copper-transporting protein and extracellular and intracellular cuproenzymes were evaluated in different brain compartments of these animals. Copper and zinc concentrations decreased significantly in different compartments of the brain of rats with experimental fibrillogenesis, while iron content did not change. According to the data of RT-PCR analysis, activities of genes of copper-transporting protein and extracellular coenzyme decreased. The expression of intracellular cuproenzyme genes and the content of SOD1 protein did not change, SOD1 activity in the cytosol decreased, and active SOD1 was detected in the mitochondrial intermembrane space. The relationship between fibrillogenesis and copper metabolism is discussed.

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.

CTR1 gene (SLC31A1 according to Entrez data base) product is the main candidate for the role of eukaryotic copper importer, whose tissue-specific function is still unclear. In this research steady state CTR1-mRNA level was measured with semiquantitative RT-PCR analysis and compared with copper status in rat organs, in which copper metabolism is changed during development (liver, cerebellum, choroid plexus and mammary gland). It has been shown that CTR1 gene activity correlates with the rate of both intracellular and extracellular copper-containing enzymes formation. In mesenchymal origin cells of newborns the CTR1 gene activity decreases when high copper concentrations in cell nucleus is reached. According to phylogenetic analysis CTR1 has the most conservative transmembrane domains 2 and 3 (TMD), containing 7 amino acid residues able to coordinate copper atom. A model of cuprophylic channel has been proposed, which is formed by TMD2 and TMD3 in homotrimeric CTR1 complex. In this model copper is transported through the channel to cytosolic C-terminal motif His-Cys-His, which ability to coordinate Cu(I) was assessed by molecular modeling (MM+, ZINDO/1). Theoretical possibility of copper transfer from His-Cys-His CTR1 C-terminal motif to cytosolic Cys-X-X-Cys Cu(I) chaperon sites has been shown. The role of CTR1 in copper metabolism as copper donor and acceptor is discussed.

This work elucidates the role of ceruloplasmin (Cp): the main soluble copper containing glycoprotein, in newborn infant copper metabolism. An over 3-fold drop of Cp and copper concentration in samples of skimmed milk from 2 to 5 days of lactation was demonstrated. It has been shown that [125I]Cp of the breast milk is discovered in the blood plasma after its was administered per os to 6-day old rats. In the body, [125I]Cp was specifically bound to cells and lost copper ions. Alternatively, after copper metabolism change to adult type, milk [125I]Cp isn't carried from gastrointestinal tract to the blood. During 8 days, newborn rats were fed with baby formula and we have found that in this case changing of the copper metabolism type takes place earlier in contrast to the litter fed by females. Transcription of the Cp gene activated in liver, Cp and the copper ions concentration in the blood plasma increased proportionally, whereas in liver the copper concentration is decreased. In the brain, changes typical for adult copper metabolism, were not discovered. But in cerebrospinal fluid copper and Cp concentration sharply increased. The role of milk Cp in controlling the copper balance in newborn rats and the tissue-specific mechanism of Cp gene activity regulation are under discussion.

Expression of two copper-transporting P1-type ATPases (ATP7A and ATP7B), the CTR1 protein, a high-affinity copper transporter, and ceruloplasmin (CP), a copper-containing ferroxidase. The level of mRNA of these proteins was determined by RT-PCR analysis, the distribution of polypeptides encoded by these genes was determined by immunoblotting, and the type of cells expressing these genes was identified immunohistochemically. It was found that the major product of CP gene in the brain is cell membrane-bound CP. Secretory CP, whose molecule contains the greatest number of weakly associated copper atoms, is synthesized in the vascular plexus. CTR1 mRNA is evenly distributed in the brain; however, its content is twice higher in the vascular plexus. The Atp7a gene is active in all brain sections, whereas the Atp7b gene is active only in the hypothalamus. The membrane-bound CP is expressed in glial cells of all types and in ependyma cells. ATP7b and ATP7a are expressed predominantly in ependyomyocytes and neutrons, respectively. The organization of copper transport in mammalian brain is discussed.

Biosynthesis of ceruloplasmin, a copper-containing glycoprotein, which plays an important role in copper transfer and bidirectional iron transport in vertebrates, was studied in 7-day old rats characterized by the embryonic type of copper metabolism. In addition to the liver, copper is synthesized in the lungs, brain, and kidneys. The appearance of labeled ceruloplasmin in the blood flow coincides with the time of liberation of de novo synthesized ceruloplasmin from the liver. [14C]-Ceruloplasmin is absorbed from the blood flow by cells of the heart, lung, and kidneys and binds to erythrocytes. The polypeptide ceruloplasmin chain does not enter the brain cells from the blood flow. Immunoreactive polypeptides of the ceruloplasmin receptor were found using immunoblotting in plasma membranes of the heart, liver, kidneys, and erythrocytes, rather than in those of the brain. It was shown by reverse transcription coupled with PCR using selective primers these cells contain molecular forms of ceruloplasmin mRNAs programming the synthesis of both secretory ceruloplasmin and ceruloplasmin connected with the plasma membrane via the glycosyl phosphatidylinositol anchor. After transition to the adult type of copper metabolism, the blood serum contents of copper and ceruloplasmin sharply increase, while the content of CP in the cerebrospinal fluid, as measured according to the oxidase and antigen activities, and copper concentration, as determined by atom-absorption spectrometry, remain low. Ontogenetic features of the system ensuring the copper homeostasis in mammals are discussed.

Using the immunoblotting method, the synthesis of two copper-transporting P1-type ATPases, ATP7A (a candidate for the product of the Menkes disease gene) and ATP7B (presumed product of the Wilson disease gene), in the yolk sac cells of rat embryos at days 11 and 20 of embryogenesis was demonstrated. Concomitantly, yolk sac cells produce ceruloplasmin, a soluble copper-transporting glycoprotein, a proportion of which in secreted proteins progressively diminishes, attaining 5.2% at day 11 and 3.1% at day 20 of development. At different stages of embryogenesis, yolk sac cells synthesize two molecular forms of [14]C-ceruloplasmin, one of which is secreted towards the embryo, whereas the other, towards the decidual membrane. Two forms of ceruloplasmin secreted in polar directions differ in the rate of secretion. The role of the yolk sac as a key organ controlling the delivery and secretion of copper in the embryo during the postimplantation period is discussed.

The interaction was studied of ceruloplasmin (Cp, EC 1.16.3.1), a copper-containing plasma protein, with two synthetic peptides P15 and P16 whose structures correlate with those of the noncytosolic regions of the copper transfer P1 type ATPase (ATP7A), apparently encoded by the Menkes disease gene (Atp7a). Pentadecapeptide P15 and hexadecapeptide P16 were synthesized using the solid phase method. They correspond to fragments of two extracellular loops ATP7A, of which one loop is apparently involved in the copper ion transfer (P16) whereas the other is not (P15). The protein footprinting showed that P16 binds to a fragment of the ceruloplasmin domain 6. Kinetics of the ceruloplasmin-P16 binding was studied by affinity chromatography on P16 immobilized on a macroporous disk, and the Kd value (1.5 x 10(-6) M) of this interaction was determined. The ATP7A involvement in the copper ion transfer to nonhepatocyte cells is discussed.

The oxidase activity of ceruloplasmin (Cp, EC 1.16.3.1), the content of immunoreactive Cp and copper ion concentration were measured in the serum of eight day-old rats receiving either breast feeding (control group) or commercial nutritive mixture which has been recommended for the newborn children beginning from zero age (experimental group). It was shown that the artificial feeding caused almost 3-fold increase of Cp oxidase activity and copper content in the serum when compared to age-matched controls. No changes in the copper content per Cp molecule were observed. Dot-hybridization of the total liver polyribosomal RNA with Cp [32P]cDNA showed that the increased Cp level in the blood of the rats of experimental group correlated well with the level of expression of Cp gene. The copper content in the liver of experimental rats was two times lower that in control animals while no differences was found in the brain copper content between two groups of rats. The role of the regulation of Cp gene expression in the lactating mammary gland and of milk Cp in the copper homeostasis in the newborn body is discussed.

In chase experiments, we followed the distribution of [125I]-ceruloplasmin prepared from human breast milk orally administered to young rats. Experiments were conducted using six-day-old rat pups (the embryonic type of copper metabolism) or 35-day-old ones (the adult type of copper metabolism). Using the technique of rocket immunoelectrophoresis, we have demonstrated that in six-day-old rats [125I]-ceruloplasmin was transferred from the gastrointestinal tract to the bloodstream and could be detected there over a period of 4 h. In 35-day-old rats, milk ceruloplasmin was digested in the upper part of the intestinal tract. The dynamic aspects of the distribution of labeled milk ceruloplasmin in the body of six-day old rats over a period of 4 h point out that, under the conditions of embryonic copper metabolism, it can serve as a transporter of copper ions to extrahepatic organs. We discuss the role of milk ceruloplasmin in copper metabolism in mammals during the neonatal period.

Expression of two copper-transporting P1-type ATPases (ATP7A and ATP7B), the CTR1 protein, a high-affinity copper transporter, and ceruloplasmin (Cp), a copper-containing ferroxidase was studied. The level of mRNA of these proteins was determined by RT-PCR analysis, the distribution of polypeptides encoded by these genes was determined by immunoblotting, and the type of cells expressing these genes was identified immunohistochemically. It was found that the major product of Cp gene in the brain is the cell membrane-bound Cp. Secretory Cp, whose molecule contains the greatest number of weakly associated copper atoms, is synthesized in the choroid plexus. CTR1 mRNA is evenly distributed in the brain; however, its content is twice higher in the vascular plexus. TheAtp7agene is active in all brain regions, whereas theAtp7bgene is active only in the hypothalamus. The membrane-bound Cp is expressed in glial cells of all types and in ependyma cells. ATP7B and ATP7A are expressed predominantly in ependymyocytes and neurons, respectively. The organization of copper transport in mammalian brain is discussed. [ABSTRACT FROM AUTHOR]

In vivo biosynthesis of ceruloplasmin (Cp), a copper-containing glycoprotein, which plays an important role in copper transfer between organs and bidirectional iron transport in vertebrates, was studied in 7-day old rats, which are characterized by the embryonic type of copper metabolism. In addition to the liver, Cp is synthesized in the lungs, brain, and kidneys. In “pulse-chase” experiments it was demonstrated that [14C]-Cp appearance in the blood coincides with the secretion of de novo synthesized Cp from the liver. [14C]-Cp is taken up from the blood stream by cells of the heart, lung, and kidneys and binds to red blood cells, while Cp polypeptide chain is not taken up by the brain cells. Immunoreactive polypeptides of the Cp receptor were found using immunoblotting in plasma membranes of the heart, liver, kidneys, and red blood cells, rather than in the brain. Using the RT-PCR method with selective primers, it was shown that these cells contain molecular forms of Cp-mRNAs programming the synthesis of both secretory Cp and Cp bound to the plasma membrane via a glycosyl phosphatidylinositol anchor. After switching to the adult type of copper metabolism, the blood serum contents of copper and Cp sharply increase, while the Cp content in the cerebrospinal fluid, as measured according to the oxidase and antigen activities, and copper concentration, as determined by atomic absorption spectroscopy, remain low. Ontogenetic features of the system ensuring the copper homeostasis in mammals are discussed. [ABSTRACT FROM AUTHOR]