[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.

Identification of species Leucochloridium paradoxum and L. perturbatum (Trematoda) based on rDNA sequences.

The full nucleotide sequences of DNA ribosome cluster of Leucochloridium paradoxum Carus, 1835 and L. perturbatum Pojmanska, 1967 were obtained. rDNA was extracted from 40 isolates of Leucochloridium sp. and analyzed using specific primers. The intraspecific genetically identity of morphologically detected L. paradoxum and L. perturbatum sporocysts was proven. A noticeable interspecific divergence between L. paradoxum and L. perturbatum was indicated. Using rDNA genotyping a case of double infection of snail Succinea sp. with L. paradoxum and L. perturbatum sporocysts was detected.

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.

[Molecular genetic analysis of trematodes of the genus Leucochloridium dwelling in the territory of Leningrad Province].

The color of the broodsac sporocyst traditionally serves as the main taxonomic criterion for distinguishing of trematodes of the genus Leucochloridium. Broodsacs of L. paradoxum (Cams, 1835) are green, while broodsacs of L. perturbation (Pojmanska, 1969) are brown. We used molecular genetic analysis of sporocyst rDNA for verifying the accuracy of the mentioned morphological criteria. Trematode infected snails Succinea sp. were collected in Vyritsa and Lyuban (Leningrad Province, Russia). Nucleotide sequences of L. paradoxum (n = 18) and L. perturbatum (n = 10) rDNA including transcribed spacers (ITS1 and ITS2) and 5.8 S rRNA gene were received, rDNA fragments of Leucochloridium sp. sporocysts of the same color were identical. The difference in the ITS1 (2.6%) and ITS2 (6.7%) between sequences of L. paradoxum and L. perturbatum was revealed. Specific nucleotide sequences are deposited at the GeneBank.

[Expression of genes encoding defense factors in the snail Planorbarius corneus (Gastropoda, Pulmonata) infested with trematodes].

Because many species of gastropods are intermediate hosts for trematodes, these molluscs are often used as model-organisms in the studies of invertebrate immune system. Revealing of the ways in which the defense factors functioning became possible due to the use of the methods of molecular biology. Contemporary molecular methods allow analyzing the defense factors allocations and levels of their expression. We investigated the expression of genes encoding defense factors in gastropods by the example of the snail Planorbarius corneus from water bodies of the Leningrad Oblast under infestation with trematods. The snails naturally infested with the parthenites of trematode species belonging to the families Strigeidae, Notocotylidae, Plagiorchiidae, and Schistosomatida were used as the experimental sample. Uninfested snails were used as a control sample. Several genes encoding the factors, which have been recently found involved in the anti-trematode defense reactions in pulmonates, were chosen, namely fibrinogen-related protein, C-lectin, calcium-binding protein, and cystatin-like protein. The genes' expression was analyzed on total mRNA samples by the reverse transcription with the polymerase chain reaction. It was shown than expression levels of the genes under consideration are different in uninfested snails and in the snails infested with different trematode species. Thus, in the mollusks infested with the parthenites of Cotylurus sp. and Bilharziella polonica, the expression levels of the genes of all factors under study were increased, while in the infested Notocotylus sp. n Plagiorchis sp., only expression levels of C-lectin and cystatin-like protein were increased. Results of the expression analysis confirm the role of hemocytes and cells of hepatopancreas in the production of humoral defense factors. In the snails infested with trematodes, the expression levels of C-lectin and calcium-binding protein genes are increased in haemocytes, while the genes of fibrinogen-related and cystatin-like proteins are activated in the hepatopancreas. Our data also confirm the role of the factors examined in the anti-trematode defense reactions in pulmonates.

[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.

[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.

[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.

[Transformation of bone marrow cells in rodents by recombinant plasmid pBRSV]. / Transformatiia kletok kostnogo mozga gryzunov rekombinantnoi plazmidoi pBRSV.

Bone marrow cells (mouse strain CBA/Ca and Syrian hamster cells) were transformed with pBRSV DNA containing T-antigen of the SV40 virus. The SV40 T-antigen in transformed cell was detected in 0.5% cases by immunofluorescence with specific antibodies. Extrachromosomal localization of recombinant DNA was shown by means of retransformation of E. coli cells with cytoplasmic spleen DNA from mice previously injected intravenously the transformed bone marrow cells.

[Autonomous replication of plasmid pBR322 containing a mitochondrial DNA fragment of animal origin in the cells of bacteria mutant for DNA-polymerase I]. / Avtonomnaia replikatsiia plazmidy pBTR322, soderzhashchei fragment mitokhondrial'noi DNK zhivotnogo proiskhozhdeniia, v kletkakh bakterii, mutantnykh po DNK-polimeraze I.

Escherichia coli K-12 p108(polA6), p3478(polA1) and KS55(polA12 ts) strains were transformed by recombinant DNA consisting of pBR322 plasmid and BamHI fragment A of rat liver mtDNA containing the origin. For all the strains tested, it was shown that the cells containing hybrid molecules were able to grow on the selective medium under the conditions when pBR322 replicon is not functional. The existence of mtDNA insertion in hybrid molecules was confirmed by electrophoresis and colony hybridization with the 125I-mtDNA. Thus, the ability of a vector containing plasmid replicon and the mitochondrial origin to replicate under the conditions nonpermissive for stable replication of plasmid DNA alone, was demonstrated for the first time.

[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.

[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.

[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.

[The possibility of the incorporation of macromolecules, including exogenous DNA, into the germ cells of male mice. The liposome method and Ca-P coprecipitation method]. / K vozmozhnosti inkorporirovaniia makromolekul, vkluchaia ékzogennuiu DNK, v polovye kletki samtsov myshei. Liposomnyi metod i metod Ca-P kopretsipitatsii.

Liposomes loaded with FITC-labeled albumin in the presence of PEG-1,500 are actively sorbed on the membranes of mature spermatozoa and remain attached even after thorough washing. Immature sperm cells are able to incorporate alien DNA carried by liposomes. In contrast, the mature spermatozoa could not incorporate plasmid DNA loaded with positively charged liposomes. Chlortetracycline in Ca-P coprecipitate crystals is tightly fixed in the postacrosomal region of mature sperms. Intensity of staining of chlortetracycline is stimulated by DNA load in Ca-P coprecipitate as well as by DMSO or EDTA. The method of Ca-P coprecipitation could not provide for plasmid DNA incorporation into taure sperms. Foreign DNA incorporation in postacrosomal regions of sperm heads seems quite possible in experiments with dimethylsulphoxide (DMSO).