[Transthyretin gene V30M, H90N, and del9 mutations in cardiomyopathy patients from St. Petersburg].

A search of transthyretin (TTP) gene mutations was conducted in patients with cardiomyopathies from St. Petersburg. Mutations H90N, V30M, G47A, and deletion (del9) of nucleotides GACTTCTCC in position 6776 from the start codon of the TTP gene (in position 98782 according to reference sequence AC079096 (NCBI) was found. The H90N mutation in the third exon of TTP gene was detected in a son of a cardiomyopathy patient and in his mother, which lacked any clinical manifestations. Mutations V30M and G47A in exon 2 of TTP gene were found in heterozygous and homozygous state, respectively, in one of the probands. Deletion (del9) was revealed in a patient with cardiomyopathy and in his two daughters from different marriages, who had no clinical manifestations of the disease. All the mutations revealed in this study were previously identified in other populations.

[The study of preimplantation development of mice embryos labelled with green fluorescent protein gene (EGFp)].

One of the crucial problems of developmental biology is the study of mechanisms of regulation of gene expression in early embryogenesis. Here we studied dynamics of mosaic appearance of a marker fluorescent protein in in vitro developing mice embryo derived from zygotes after microinjections to male pronuclei of cloned DNA fragment carrying EGFP under control of different promoters. Main attention was paid to initial stages of development, when structural rearrangements and reprogramming of both parental genomes, activation of zygotic genes, and control of development by embryo genome take place.

Role of the C-terminal fragment of human transthyretin in abnormal fibrillogenesis.

Polypeptide chain fragments of recombinant transthyretin (TTR) with leucine-55 substituted by proline (L55P), which are involved in abnormal fibrillogenesis of this protein, were studied. No fibrils were produced in purified preparations of TTR(L55P) under the optimum conditions for fibrillogenesis but in absence of protease inhibitors. The ability of TTR for fibrillogenesis was lost because of a limited proteolysis resulting in detachment of the TTR polypeptide chain C-terminal fragment of approximately 18 amino acid residues in length. This proteolysis seemed to occur with involvement of a bacterial serine endopeptidase sohB (EC 3.4.21), which was identified in TTR preparations by the MALDI-TOF method. The presence of the C-terminal fragment of the TTR polypeptide chain seems to be crucial for production of abnormal fibrils.

[The role of quarternary structure in fluorescent protein stability].

The stability of fluorescent proteins (FPs) is of great importance for their use as reporters in studies of gene expression, protein dynamics and localization in cell. A comparative analysis of conformational stability of fluorescent proteins, having different association state was done. The list of studied proteins includes EGFP (monomer of green fluorescent protein, GFP), zFP506 (tetramer GFP), mRFP1 and "dimer2" (monomer and dimmer of red fluorescent protein), DsRed1 (red tetramer). The character of fluorescence intensity changes induced by guanidine hydrochloride (GdnHCl) of these proteins differs significantly. Green tetramer zFP506 has been shown to be more stable than green monomer EGFP, red dimmer "dimer2" has been shown to be less stable than red tetramer DsRed1, while red monomer mRFP1 has been shown to be practically as stable as tetramer DsRedl. It is concluded that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stability.

[Expression of marker genes in muscle fibers after transfection in vivo, mediated by lactoferrin]. / Ekspressiia markernykh genov v myshechnykh voloknakh posle transfektsiiin vio, oposredovannoi laktoferrinom.

The capacity of milk iron-transporting human protein lactoferrin (LF) to deliver genetic constructions into cells was studied in an effort to correct hereditary defects. The purified LF and LF conjugates containing either polylysine (C-1) or both polylysine and ficoll (C-2) were bound to plasmid DNA. These complexes were injected into mouse muscles, and the expression of the marker genes was tested immunochemically. Mice were transfected with either pDMD1 plasmid carrying a full-size cDNA for human dystrophin gene or pCMVLacZ plasmid carrying the gene of bacterial beta-galactosidase. The marker gene expression was detected in the muscular fibers. The dystrophin-positive muscular fibers (DPMF) were revealed in mdx mice (a model of Duchenne's dystrophy) in the regions of administration and in muscles of the other limbs. beta-Galactosidase activity was revealed only in the injected muscles. The highest amount of DPMF (9%) was recorded in mice who received the complex of DNA with nonmodified LF. Specific LF-mediated human transfection as a means of stimulating the receptor-mediated endocytosis of genetic constructions and addressed gene transfer to human muscles are 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.

Comparative analysis of the molecular heterogeneity of ceruloplasmin from human blood and breast milk.

The content of ceruloplasmin (Cp) was determined in 96 samples of human breast milk using rocket immunoelectrophoresis and measurement of Cp oxidase activity. The concentration of immunoreactive Cp in milk decreased about 9 times during the first 20 days of lactation while the specific oxidase activity decreased only 4 times. Two-dimensional electrophoresis of purified milk Cp before and after its treatment with chelating agents showed that copper atoms in milk Cp are more sensitive to EDTA treatment that those in blood Cp. The comparison of the different lectin-binding ability of blood and milk Cp's revealed a difference in the composition of their carbohydrate chains. The mechanisms controlling the uptake of copper ions by newborns at the level of the expression of the Cp-encoding gene in the mammary gland of the mother are discussed.

Reconstitution of the intercellular transfer pathway of the peptide moiety of ceruloplasmin in mammals.

According to rocket immunoelectrophoresis, the antigenic properties of the ceruloplasmin (Cp) receptor present on the surface of human fibroblasts are similar to those of the Cp receptor of the erythrocyte plasma membrane. Using antibodies to Cp receptor, it was demonstrated that Cp binds to the surface of fibroblasts only via the Cp receptor. Ceruloplasmin was labelled with radioactive iodine and its binding to cultured human HT-1080 fibroblasts was studied; at saturating concentrations [125I]Cp interacts with cell surface of fibroblasts (but not hepatocytes) with high affinity (Kd = 80 nM). Subsequent to specific binding fibroblasts absorb [125I]Cp and in about 120-150 min start to release it into the incubation medium. Two fractions of released Cp are clearly detected by non-denaturing polyacrylamide gel electrophoresis. The relative mobility of one of these fractions corresponds to apo-Cp and the other has lower mobility than native Cp. The molecular weight of released [125I]Cp is changed insignificantly. Released Cp does not bind again to the fibroblast surface but is bound, absorbed, degraded, and secreted by hepatocytes. The molecular mechanism of cell-specific transfer of Cp in the human body is discussed and possible functions of this mechanism in copper absorption, metabolism, and excretion in mammals are considered.

[Regulation of biosynthesis of molecular forms of ceruloplasmin in rat ontogenesis]. / Reguliatsiia biosinteza molekuliarnykh form tseruloplazmina v ontogeneze krys.

Ceruloplasmin (CP) expression at the level of formation of its molecular forms in the liver and other rat organs during ontogenesis has been studied. The synthesis of CP commenced on day 15th of embryogenesis and coincided in time with the completion of formation of the fetal liver. This period of rat embryo development was marked by the appearance of two molecular forms of newly formed CP, one of which was secreted in vitro by isolated fetal liver, while the other one was found in the membrane fraction of extrahepatic embryonic tissues. The proportion of CP among the total protein of these fractions increased up to term, correlating with the gain in the embryo weight. The relative content of CP secreted by the livers of newborn animals remained constant up to day 9th of postnatal life and then increased with the age up to approximately 1.5 months. At the same time, the serum concentration of CP remained constant up to day 19th of postnatal life. The increased rate of secreted CP synthesis in the liver during ontogenesis coincided with the appearance of a newly formed non-secreted membrane-bound form of CP in hepatocytes. The ontogenetic dynamics of synthesis of various molecular forms of CP in the liver correlated with the results of immunoblotting of CP polypeptides in Golgi membranes isolated from the livers and the livers and the liver of a 30-day-old rat.

[Biosynthesis and secretion of ceruloplasmin by rat mammary cells during lactation]. / Biosintez i sekretsiia tseruloplazmina kletkami molochnoi zhelezy v period laktatsii u krys.

Some peculiarities of rat milk ceruloplasmin (CP) biosynthesis have been analysed. Electrophoretic and immunoelectrophoretic data suggest that rat milk contains up to 20 mg/100 ml of CP. Rat milk CP is represented by a single molecular form whose molecular mass, enzymatic activity and antigenic properties are fully identical with those of serumal CP. According to the dot hybridization date, the CP-mRNA content in rat mammary cells is about ten times as low as that in adult rat liver. Analysis of polyribosomal RNA by blot hybridization of RNA-cDNA revealed that rat mammary CP-mRNA is represented by a single molecular form, 3.5 kb in length, whose molecular mass is identical with that of the predominant CP-mRNA of rat liver. The dynamics of secretion of the de novo synthesized CP into rat milk indicates that the CP synthesis and secretion occur independently of the serumal CP. In vitro and in vivo pulse labelling studies demonstrated that rat milk CP is synthesized and secreted by rat mammary cells.

[Biosynthesis of two molecular forms of ceruloplasmin in rat liver and their polar secretion into the blood stream and bile]. / Biosintez dvukh molekuliarnykh form tseruloplazmina v pecheni krysy i ikh poliarnaia sekretsiia v krovotok i v zhelch'.

Biosynthesis and secretion of ceruloplasmin (CP) in rat liver has been studied in order to elucidate its role in the distribution, transport and excretion of copper in the body. The kinetics and topography of CP synthesis, intracellular transport and secretion were followed using in vivo pulse-chase experiments. It was found that the newly formed CP was firmly bound to endoplasmic reticulum membranes in the hepatocytes. Liver slices incubated in vitro with [35S]methionine were characterized by a two-stage release of [35S]CP into the medium. After 30-min incubation the medium contained 200 kDa CP-like protein, while after 120 min the secretion of 130 kDa CP was demonstrated. The pulse-labelling experiments with [35S]methionine in rats with catheters inserted into the carotid artery and the common bile duct revealed the polar secretion of two distinct CP species differing in molecular structure and secretion rate. The slowly secreted CP isoform is the authentic serum CP, while the rapidly secreted CP is the specific biliary CP. The physiological function of biliary CP and its role in the pathogenesis of Wilson's disease are discussed.

[Properties of silver-containing rat ceruloplasmin]. / Svoistva tseruloplazmina krysy, soderzhashchego serebro.

Ceruloplasmin (Cp) was isolated from the sera of albino rats fed with silver nitrate (60 mg/kg of body weight). The oxidase activity of the enzyme was sharply decreased, while its concentration in the blood (as assayed immunologically) was slightly lower than in controls. The drop in the oxidase activity was caused by the replacement of several coppers by silver ions in the Cp molecule. Ag-Cp contained about four silver atoms per 1 mole of protein, its spectrum lacking maxima at 450 and 610 nm that are typical of normal Cp. When subjected to PAAG electrophoresis, Ag-Cp displayed two bands, one of which (Ag-Cp2) had the anodic mobility of normal Cp. The other band (Ag-CpI) migrated at a slower rate. Both bands were separately subjected to SDS-PAAG electrophoresis which revealed the dissimilarities among the proteolytic fragment patterns of Ag-CpI, Ag-Cp2 and normal Cp. Both Ag-CpI and Ag-Cp2 contained peculiar fragments produced by spontaneous limited proteolysis of the native molecule. The binding of silver ions by Cp seems to alter significantly the molecule conformation, which may cause the exposure of new peptide bonds susceptible to proteolytic attack. Cp seems to participate in the binding and detoxication of heavy metals in mammals.

[Etiologic significance of hereditary deficiency of proteinase alpha1-inhibitor in the formation of respiratory diseases]. / Etiologicheskoe znachenie nasledstvennogo defitsita alpha1-ingibitora proteaz v formirovanii zabolevanii organov dykhaniia.

A study was made of the content of protease alpha 1-inhibitor and of the phenotyping of protease alpha 1-inhibitor subtypes in 666 patients with different chronic non-specific pulmonary diseases. It is concluded that pronounced deficiency of protease alpha 1-inhibitor is of importance in the formation of primary emphysema, chronic obstructive bronchitis and bronchial asthma. The clinical characteristics of obstructive pulmonary diseases marked by protease alpha 1-inhibitor deficiency have been investigated.

[Synthesis and secretion of ceruloplasmin by isolated rat hepatocytes]. / Sintez i sekretsiia tseruloplazmina izolirovannymi gepatotsitami krysy.

The synthesis and secretion of ceruloplasmin (Cp) by isolated rat hepatocytes were investigated. Cp released by liver cells appeared to have properties similar to those of the blood-circulating protein, i. e. Mr, oxidase activity, immunological specificity and the peptide set of tryptic fingerprints. The polypeptides with Mr of 130,000, 65,000, 48,000 and 18,000 were revealed in Cp isolated from the incubation medium. These results suggest the susceptibility of the single-chain protein molecule (Mr 130,000) to limited proteolysis which is accomplished by the proteases released from the cells. When fresh serum was added to the incubation medium, the proteolytic degradation of Cp proceeded at a much slower rate, which led to an increase in the content of excreted polypeptides with Mr 130,000. The secretion was strongly diminished by the addition of colchicine to the medium. The time of Cp molecule synthesis on membrane-bound polyribosomes (3.5 min) was determined.