[The changes of copper metabolism in rats fed with low- or high-calorie ration].

Copper is an essential micronutrient, because it is a catalytic and structural cofactor of enzymes that control the basic processes in all cells, and moreover it is a participant in signaling pathways. The toxic properties of copper ions, due to their chemical nature, are manifested when the cellular and/or organism systems for copper homeostasis are disturbed. Aim of the work was to study the relationships between the diet caloric and the copper status in the blood serum, the copper metabolism in the liver and white adipose tissue (WAT) of rats. MATERIAL AND METHODS: The work was performed on three groups (each n=5) of white outbred rats (average body weight 220±15 g), kept for 75 days on a standard, low-calorie (LCR) or high-calorie (high-fat) (HCR) rations. mRNA concentration was measured by qRT-PCR technology. The сeruloplasmin (CP) content was determined by the method of immune electrophoresis, immune blotting and by oxidase activity. The copper concentration was measured by atomic absorption spectrometry. RESULTS AND DISCUSSION: It has been shown that serum level of triglycerides increased in rats fed LCR. The main indicators of copper status (concentration of atomic copper, the level of holo-CP, and the content of immunoreactive CP) decreased in rats fed HCR. In the liver, none of the diets affected Cp gene expression level. In the cells of the subcutaneous fatty tissue, the concentration of both splice-forms of CP-mRNA significantly increased in rats fed LCR. In visceral adipose tissue the concentration of Cp-mRNA encoding the secretory CP did not change in LCR-rats, but the level of mRNA, encoding CP anchored to plasma membrane, dropped to almost zero as compared to the control group. There was no significant change in the level of both splice-forms of CP-mRNA in HCR-rats. The features of copper metabolism in the cells of the liver and WAT, due to the caloric content of ration, have been discussed. CONCLUSIONS: In rats' liver, the link between copper metabolism and calorie intake is manifested in changes in the expression of the CP gene at the translation level, and in white adipose tissue - at the level of transcription and post-transcriptional maturation of the pre-mRNA of this gene.

Role of Copper Dyshomeostasis in the Pathogenesis of Parkinson's Disease.

Serum concentration of copper, immunoreactive polypeptides of ceruloplasmin and its oxidase activity, and the number of copper atoms per ceruloplasmin molecule were decreased in patients with Parkinson's disease in comparison with the corresponding parameters in age-matched healthy individuals, but the ratio of apoceruloplasmin to holoceruloplasmin in patients with Parkinson's disease was similar in both groups. Treatment of blood serum with Helex 100, a high-affinity copper chelator, revealed reduced content of labile copper atoms per ceruloplasmin molecule in patients with Parkinson's disease in comparison with that in healthy controls. The mechanism underlying impaired metabolic incorporation of labile copper atoms into CP molecule is discussed as a possible cause of copper dyshomeostasis associated with Parkinson's disease.

[The nutrition role of milk ceruloplasmin].

Copper is an essential trace element for all aerobic organisms. In mammals, it is a structural and redox-based co-factor of the vital enzymes. Besides catalytic function, copper acts as a modulator of cell signaling ways. However copper ions outside the pre-organized coordination sphere can initiate the formation formation of reactive oxygen species through Fenton type reactions. Both deficiency and excess of copper lead to the development of the cardiovascular, neoplastic, and neurodegenerative diseases. In the present article the data on copper physiological functions as well as peculiarities of the mechanism of copper safe transport are briefly reviewed. Also the results of investigation of the mechanisms supporting copper homeostasis and mechanistic features of copper homeodynamics in the newborns are summarized. The data on molecular genetic mechanism of the mammary glands, which controls nutrition copper balance for newborns, are analyzed. The role of milk ceruloplasmin as the newborn's main source of nutritional copper is discussed. The quantity and quality of the baby formula copper-containing additives and their potential long-term health effects are considered.

[Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

Development of laboratory rats receiving silver-enriched ration for a long time.

We studied the effect of silver ions on the status and metabolism of copper in rats receiving Ag-diet from the first day of life and for 6 months. The effect of silver ions on copper metabolism was assessed by body weight, relative weight of organs (body weight/organ weight), oxidase activity, content of immunoreactive ceruloplasmin and copper concentration in blood serum, by the expression of copper-transporting protein genes in the liver, and copper and silver distribution in liver and brain cells. Brain functions were evaluated by open-field behavior and passive avoidance conditioning. No acute deficiency of ceruloplasmin-associated copper was observed in rats receiving silver-enriched diet starting from the early postnatal period; copper metabolism in the liver did not change, psychoemotional state and memory corresponded to the control. However, Ag-diet almost 2-fold decelerated the growth of experimental rats. We hypothesize the existence of an unknown mechanism of copper delivery to organs in rats that is activated during the early ontogeny under conditions of ceruloplasmin-associated copper deficiency.

[Mitochondrial and lysosomal pathways of death of hepatocytes of lamprey Lampetra fluviatilis L].

Mechanisms of mitochondrial and lysosomal pathways of natural cell death in lamprey hepatocytes at the spring period of prespawning migration are described. The mitochondrial pathways (release of cytochrome c from mitochondria into cytosol and activation ofcaspases) operates according to the classical scheme known for apoptosis. The lysosomal cell death pathway connected with activation of cathepsin B has been revealed quite recently in cells in pathologies, in particular at obstruction of gallbladder and bile ducts. The peculiarity of lamprey hepatocytes consists in biliary atresia (the absence both of gallbladder and of bile ducts) in liver of adult animals. Thereby the lamprey hepatocytes represent an excellent object for study of this new pathway of cell death. We have revealed a parallel development of the mitochondrial and lysosomal pathways of cell death of lamprey hepatocytes.

[Characteristics of rat ceruloplasmin from the serum of animals, which received salts of silver with food].

Abiogenic Ag(I) ions have electronic structure, similar to Cu(I) ions and can compete with Cu(I) for binding sites of proteins which transport copper from extracellular media to sites of cuproenzyme formation in the cell. Rodents receiving Ag-salts with food develop extracellular deficiency of copper associated with ceruloplasmin (Cp, the major copper-transporting protein in blood serum of vertebrates). The present work focuses on the studies of biochemical and physicochemical properties of Cp, obtained from blood serum of rats, which received AgCl with food for 4 weeks (Ag-rats). Cp-fractions from blood serum of Ag-rats (Ag-Cp) were obtained by ion-exchange chromatography with stepped gradient of NaCl. Each fraction was tested for oxidase and ferroxidase activities by direct measurement of catalytic activity in the gel, and for specific activity in holo-Cp in oxidation of chromogenic substrate. Molecular mass, electrophoretic mobility and ratio of apo- and holo-forms in Ag-Cp fractions were evaluated by immunoblotting. Ag-Cp samples did not contain products of spontaneous partial proteolytic degradation, characteristic of holo-Cp samples. Fractions of Ag-Cp and holo-Cp (from blood serum of control rats) were compared by optical spectra, tertiary structure, susceptibility to thermal denaturation, and by atomic Cu and Ag content. Ag-Cp contained 1-2% Cp, which is similar by spectral and catalytic properties with holo-Cp. [Ag]:[Cu] ration in Ag-Cp samples was about 4:1. As evidenced by circular dichroism and differential scanning calorimetric studies, the major apo-fraction of Ag-Cp lacked tertiary structure of native Cp and was significantly misfolded, which might explain its resistance to spontaneous partial proteolytic degradation.

[Regulation of ceruloplasmin gene activity in mammary gland cells].

Tissue-specific regulation of the expression of ceruloplasmin (CP) gene, which encodes major copper-containing extracellular glycoprotein was investigated. A decrease of the CP concentration associated with copper amounts in milk during the first 3 days of lactation was used as phenotypic index for evaluating the CP enzyme activity in the mammary gland. Computer analysis of mammalian CP gene promoter region has revealed conserved sequences of cis-elements, which potentially were capable of regulating the enzyme activity. It has been shown that changes in the nucleotide sequence of specific transcriptional factor binding sites located at 5'-end of CP gene were associated with disturbance of the regular downregulation of CP gene activity during lactation.

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.

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.

[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.

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.

[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.

Isolation and partial characterization of molecular forms of ceruloplasmin from human bile.

Highly purified ceruloplasmin (CP) was isolated from human bile using affinity chromatography. Biliary CP is represented by two molecular species. One of those is identical to oxidase CP from normal human serum while the other is analogous to oxidase-lacking CP specific for the serum of the carriers of Wilson's mutation with respect to immunological specificity, electrophoretical mobility and molecular mass of the large fragments from spontaneous proteolysis.

[The regulator effect of fructose-1,6-diphosphate and cyclic adenosine monophosphate on protein and RNA synthesis by isolated mitochondria]. / O reguliruiushchem vliianii fruktozo-1,6-difosfata i tsiklicheskogo adenozinmonofosfata na sintez belka i RNK izolirovannymi mitokhondriiami

Effects of fructoso-1,6-diphosphate (FDP) and cyclic 3',5'-adenosine monophosphate (cAMP) on various steps of protein biosynthesis in isolated rat liver mitochondria were investigated. It was shown that FDP repressed and cAMP depressed the incorporation of both 14C-amino acid and [3H]uridine into mitochondrial polysomes. Cyclic 2',3'-adenosine monophosphate, a physiologically inactive analog of cAMP, had no depressing effect on the polysomes formation in mitochondria. Effects of FDP and cAMP on the synthesis of mitochondrial RNA at different periods of incubation (5, 10, 30 min) were studied. It was found that FDP repressed the high molecular weight mitochondrial RNA biosynthesis and prevented the mRNA formation. cAMP derepressed the FDP effect. Rifampicin prevented the derepressing action of cAMP. The rate of protein synthesis in the translation system isolated from mitochondria was affected neither by FDP nor by cAMP. Authors concluded that in the mammalian mitochondria the repression of protein synthesis by a glycolytic metabolite (FDP) and its derepression by cAMP represented regulatory mechanism acting at the transcription level like catabolite repression-derepression in microorganisms.

[The origin of mitochondrial RNA in animal tissues. 4. Post-transcriptional RNA modification and polyribosome formation in isolated mitochondria]. / Proiskhozhdenie RNK mitokhondrii zhivotnykh tkanei. IV. Posttranskriptsionnaia modifikatsiia RNK i obrazovanie poliribosom v izolirovannykh mitokhondriiakh

The kinetics of synthesis of various mitochondrial RNA classes and their contribution to polyribosome formation was studied in isolated rat liver mitochondria and in inner membrane fraction obrained from the latter. In pulse labelling experiments it was shown that mitochondrial mRNA synthesis occurred via heavy precursor (30S) not bound to polysomes followed by maturation of polysome-bound mRNA. The time course of polysomal mRNA decay in isolated mitochondria and of protein synthesis inactivation were studied under actinomycin D blockage of mitochondrial DNA trascription. The functional organization of transcriptional units of the mitochondrial genome is discussed in relation to the data obtained.

[Molecular mechanisms of the control of ceruloplasmin synthesis by estradiol]. / Molekuliarnye mekhanizmy regulirovaniia sinteza tseruloplazmina pod deistviem estadiola.

Mechanisms of the effect of estradiol on the biosynthesis of ceruloplasmin (CP) in rat liver were studied. The radioimmunological tests revealed that estradiol caused an increase of CP concentration in plasma. The about two-fold increase of plasma CP level was accompanied by the proportional increase of the content of CP nascent chains in membrane-bound polysomes of liver. Cell-free translation of free and membrane-bound polysomes revealed that there is a two-fold increase in the relative rate of CP synthesis by membrane-bound polysomes from estradiol-treated rats, while free polysomes did not contribute to CP synthesis. The similar increase of the rate of CP synthesis was found also when RNA preparations from membrane-bound polysomes and from post-polysomal supernatant were translated in a heterologous cell-free system. Studies on the kinetics of hybridization with a specific cDNA probe showed that the increase in the rate of CP synthesis correlates to the increase of the steady-state level of CP mRNA in both membrane-bound polysomes and post-polysomal supernatant. A conclusion is made that the induction of CP by estradiol is related to the accumulation of CP-mRNA in liver cells.

[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.

[Molecular defect of ceruloplasmin synthesis and maturation in Wilson-Konovalov disease]. / Molekuliarnyi defekt sinteza i sozrevaniia tseruloplasmina pri bolezni Vil'sona-Konovalova.

The radioimmunochemical study of ceruloplasmin-synthesizing polyribosomes was carried out using bioptic liver specimens obtained from fourteen homozygous patients with hepatolenticular degeneration (Wilson--Konovalov disease) and from eight control patients with various non-hereditary diseases. The measurement of binding of 125I-antibodies to the nascent polysome-bound ceruloplasmin chains demonstrated that in control patients this protein is only synthesized on membrane-bound polysomes, while free polysomes do not contribute to the synthesis of ceruloplasmin. The majority of homozygous carriers of Wilson--Konovalov mutation (eleven of fourteen) are characterized by the involvement of free, rather than membrane-bound polysomes, in the synthesis of ceruloplasmin. This shift of ceruloplasmin synthesis from membrane-bound to free polysomes seems to be accompanied by disturbances in the cotranslational proteolytic maturation of ceruloplasmin from its biosynthetic precursors. As a result of this defect, a putative of ceruloplasmin (preproceruloplasmin) was detected in the content of Golgi complex as well as in the serum of homozygous patients. This precursor of a molecular weight 84,000 was found neither in Golgi complex, nor in the serum of control subjects.