Changes in copper metabolism in different compartments of the brain in rats with induced fibrillogenesis.

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.

Effect of a deficiency of ceruloplasmin copper in blood plasma on copper metabolism in the brain.

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.

[8-OH-DPAT modulates expression of 5-HT(1A)/5-HT(2A), 17beta-estradiol receptor mRNAs in ovariectomized rats in Porsolt test].

The influence of chronic (14 days) administration of 5-HTIA receptors agonist--8-OH-DPAT (0.05 mg/kg, s.c.) and 5-HT(1A) receptors antagonist NAN-190 (0.1 mg/kg, i.p.) alone or in a combination with 17beta-estradiol (0.5 mg on each animal, i.m.) for on depressive behavior and expression of 5-HT(1A)-, 5-HT(2A)-, 17beta- estradiol receptors mRNAs was estimated in hippocampus in adult ovariectomized (OVX) female rats. The model of depression in rats was carried out in Porsolt test. The measurement of expression of 5-HT(1A)-, 5-HT(2A)-, 17beta-estradiol receptors mRNAs in the hippocampus was performed by semiquantitative RT-PCR. In Porsolt test 17beta-estradiol in OVX rats reduced time immobility to some extent. 8-OH-DPAT alone significantly decreased time immobility in OVX rats. Chronic 8-OH-DPAT administration in a combination with 17beta-estradiol in OVX females resulted in potentiated antidepressive effect. Simultaneously, 8-OH-DPAT induced significant increase of 5-HT(1A)-, 5-HT(2A)-receptors mRNAs expression and decrease of 17beta-estradiol receptor mRNA expression in hippocampus in OVX rats as compared to the control. The data obtained indicate a close interaction of the ovary hormonal and serotonergic systems of the brain in mechanisms of depression.

[Relations between CTR1 gene activity and copper status in different rat organs].

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.

[Identification of the rat ceruloplasmin mRNA isoform putative coded of a protein localized in mitochondria].

Alternative expression of ceruloplasmin (Cp) gene, whose product, blue multicopper ferroxidase, is a neuron survival factor, was studied in the current work. Computer analysis showed that Cp-mRNA isoform, coding for 109 kDa polypeptide, can be formed as a result of the transcription from the alternative promoter in 3'-region of intron 2 of rat Cp gene. Alternative Cp form starts with 25 amino acid residues sequence, coded with intron 2 region. It is followed by amino acid sequence of the main Cp isoform starting from Gly 113. In silico data were experimentally confirmed using RT-PCR. It was demonstrated that the predicted mRNA was generally localized in liver and brain cells of adult rats. Direct sequencing of the obtained PCR-product showed the entire coincidence of the real and predicted mRNAs. It was in vitro showed that approximately 110 kDa Cp-like protein was completed and accumulated in the absence of mitochondria. This protein is transferred into the isolated mitochondria in the reconstructed system. Transport is energy-dependent, it is not accompanied with the shortening of Cp polypeptide length and needs the presence of cytosolic factors. Probably import is determined by the inner protein mitochondria import signal with amino acid sequence KVVYREFTDSTFRE, located in 756-769 region of mature Cp. Possible role of Cp in iron metabolism in mitochondria is under discussion.

[Milk ceruloplasmin as a physiological source of nutritional copper in early ontogenesis in mammals].

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.

[In vivo expression of copper transporting proteins in rat brain sections].

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.

The revelation of expressing region in the processed ceruloplasmin gene in human genome by biocomputational and biochemical methods.

ANNOTATION: Translation in all open reading frames (ORF) of human ceruloplasmin (Cp) pseudogene revealed the only translating sequence of 984 nucleotides. The amino acid sequence contains a signal peptide for mitochondrial protein import at N-terminus. The predicted protein without taking the signal peptide into consideration has 92% identity to the corresponding Cp fragment. It contains 20 amino acid substitutions, 8 of them are significant. There is His-X-His motif in the center of a molecule that is typical for copper containing oxidases. Potential copper-binding site appears as a result of the substitution P923H along human Cp sequence. Cp pseudogene transcription product was found in the cultured human cell lines HepG2 and HuTu 80 using RT-PCR strategy. Cp polypeptides with molecular weight of nearly 30 kDa were found in mitochondria of HuTu 80 cells. The possible biological role of mitochondrial Cp is under discussion.

[Mitochondrial ceruloplasmin of mammals].

Using immunoblotting method it was found out that ceruloplasmin (Cp) polypeptides are revealed in mitochondria of rats, isolated from brain, liver, testicles and mammary gland. Cp is localized in matrix and inner membranes of mitochondria. Its molecular weight corresponds to the non-glycosilated form of the protein. Computer analysis did not reveal any sequences homologous to the signal peptide for mitochondria protein import (SPMPI) in the rat chromosomal Cp gene. However the analysis of homologous to Cp sequences, presented in databases, detected SPMPI in the human processed Cp pseudogene. Cp pseudogene region of 984 nucleotides long is translated in the only reading frame to the polypeptide of 328 a.a. long including 66 a.a of SPMPI at N-terminus. The predicted protein with the exception of SPMPI is identical to the corresponding Cp fragment at 92%, it has 20 amino acid substitutions, 8 of which are significant. His-X-His motif, typical for copper containing ferroxidases, is located in the centre of a molecule. Potential copper-binding site appears as a result of proline to histidine substitution at 923 position along Cp sequence. The product of Cp pseudogene transcription was detected in the human cultured cells of HepG2 and HuTu 80 cell lines using RT-PCR strategy. 30 kDa polypeptide that interacts with human Cp antibodies was found in mitochondria of HuTu 80 cells. The possible biological role of mitochondrial Cp is under discussion.

[Age-related features of ceruloplasmin biosynthesis and distribution in rats]. / Vozrastnye osobennosti biosinteza i raspredeleniia tseruloplazmina v organizme krys.

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.

Regulation of ceruloplasmin gene in mammals.

A site of rat DNA (about 1800 b. p.) adjacent to the first ceruloplasmin gene contains, apart from regulatory sequences common for all eukaryotic promotors, cis-elements, which are potential binding sites for soluble nuclear receptors of some hormones. Sequences characteristic of genes expressed in liver cells and mammary gland cells during lactation were detected. Full-length fragment of this locus of ceruloplasmin gene (1800 b. p.) was synthesized by PCR and used in gel shift experiments. It was found that soluble proteins extracted from purified nuclei of mammary gland cells during lactation and from the liver of adult and newborn rats, contain proteins specifically interacting with the PCR product. A fragment of chromosome gene containing exons encoding the central part of rat ceruloplasmin was cloned in pTZ19 bacterial vector. Gel shift assay showed that the cloned fragment contained binding sites for specific transcription factor YY1, whose level in nuclear protein fractions varied during ontogeny (according to immunoblotting data). Monoclonal antibodies detected protein YY1 in the complex of cloned DNA-nuclear proteins. Possible mechanisms of tissue-specific regulation of ceruloplasmin gene varying during ontogeny are discussed.

[The role of the yolk sac in copper metabolism during rat embryogenesis]. / Rol' zheltochhogo meshka v metabolisme medi v c'mbriogeneze krys.

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.

[Identification of a fragment of ceruloplasmin, interacting with copper-transporting Menkes ATPase]. / Identifikatsiia uchastka tseruloplazmina, vzaimodeistvuiushchego s med'transportnoi ATP-azoi Menkesa.

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.

Isolation and partial characterization of cDNA clone of human ceruloplasmin receptor.

An individual clone, presumably carrying a 3 bp fragment of ceruloplasmin receptor cDNA was isolated from the expression library of human placenta cDNA using polyclonal specific antibodies to ceruloplasmin receptors. EcoR1-hydrolysate of isolated DNA was cloned in a pTZ19 bacterial vector and sequenced in the forward and reverse direction. The comparison of the revealed sequence with known sequences of human genome revealed its high similarity to ceruloplasmin cDNA.

[Effects of infant formula feeding on the distribution of copper in the body of 8-day-old rats]. / Vliianie iskusstvennogo vskarmlivaniia na raspredelenie medi v organizme 8-dnevnykh krysiat.

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.

[A comparative study of the transport dynamics of the peptide moiety of the milk ceruloplasmin molecule in the body of rats with embryonic and adult types of copper metabolism]. / Sravnitel'noe izuchenie dinamiki peremeshcheniia peptidnoi chasti molekuly tseruloplazmina moloka v organizme krys pri émbrional'nom i vzroslom tipakh metabolizma medi.

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.