[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.

[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.

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.

[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.

[Expression of recombinant actin 5C from Drosophila in the methylotrophic yeast Pichia pastoris].

A system for actin expression in cells of yeast Pichia pastoris was constructed. Drosophila actin 5C, by 90% homologous to beta-actin of higher eukaryotes, was used as a target protein. To improve the procedures of target protein biosynthesis in yeast cells and of extraction and purification of recombinant actin the fusion protein GFP-actin 5C, having fluorescence protein GFP as a reporter part, was expressed and purified. The dimensions and resistance of yeast cells producing recombinant actin were characterized. It was shown that the size and form of cells depended on the accumulation of recombinant protein. The purified fusion protein was used for obtaining polyclonal antibody for testing recombinant actin.

[Isolation of stable human ceruloplasmin and its interaction with salmon protamine].

An interaction was discovered between ceruloplasmin (CP, a ferro-O2-oxidoreductase, EC 1.16.3.1), a copper-containing protein of human blood plasma, and salmon protamine (PR), a cationic polypeptide of vertebrates that provides a compact structure of spermatozoid DNA. Addition of PR to CP at a molar ratio of 2: 1 decreases the CP electrophoretic mobility. Two types of CP binding centers for PR were determined: two centers with a high (Kd1 of 5.31 x 10(-7) M) and four centers with a low affinity (Kd2 of 1.56 x 10(-5) M). PR was shown to form complexes with CPs of various animal species. The CP-PR complex dissociates at an increased ionic strength (0.3 M NaCl), at pH decreased below 4.7, or in the presence of added polyanions (DNA, lipopolysaccharides, or heparin) and/or polylysine, which indicates the electrostatic nature of the interaction. The CP-PR interaction increased 1.5-fold the rate of CP-catalyzed oxidation of Fe2+. The preliminary treatment of blood plasma with arginine-Sepharose and heparin-Sepharose (to remove the blood coagulation factors) and affinity chromatography on PR-Sepharose helped isolate the practically unproteolyzed monomeric CP in 90% yield; it remained stable for more than two months at 37 degrees C. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

[Room temperature phosphorescence of amorphous aggregates and amyloid fibrils resulting from protein misfolding].

Using actin, alpha-lactalbumin and insulin as examples, it was shown that the formation of amorphous aggregates of proteins and amyloid fibrils leads to an increase in the rigidity of tryprophan and tyrosine residues micro-environment and, consequently, to the appearance of tryptophan (tyrosine) room temperature phosphorescence (RTP). RTP was used for examining a slow intramolecular mobility of native (G-, F-form) and inactivated (I) rabbit skeletal muscle actin during the process of GdnHCl induced protein unfolding. This method made it possible to confirm that an essentially unfolded intermediate precedes the formation of inactivated actin. It has been found that the kinetic intermediate generated at the early stage of protein denaturation has no tryptophan RTP, suggesting a high lability of its structure. Symbate changes of integral intensity (relative quantum yield) and the mean lifetime of RTP during the U*-->I transition suggest a gradual increase of the number of monomers incorporated in the associate (U*-->11...-->In...-->I15), which is accompanied by an increase of protein structural rigidity. The rate of inactivated actin formation (I-->I15) is shown to increase with the increase of protein concentration. It is shown that, no matter what method of inactivation was employed (1--2 M GdnHCl or 3.0-3.5 M urea, Ca2+ removal, incubation at 70 degrees C, refolding from completely unfolded state by dialysis from 8 M urea or 6 M GdnHCl), actin transition to the inactivated state is accompanied by a significant increase in both integral intensity and the mean lifetime of RTP, suggesting the rigid structure of inactivated actin. It is shown that the lifetime of inactivated actin RTP does not depend on GdnHCl concentration within the limits from 0 to 4 M. On using insulin and alpha-lactalbumin as examples, it is shown that RTP can be used in studies of fibrillogenesis and properties of amyloid 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.

[Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene]. / Ispol'zovanie gena supressornoi tRNK dlia ispravleniia nonsens-mutatsii v gene distrofina.

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.

[Clinical polymorphism and ceruloplasmin variants in hepatolenticular degeneration]. / Klinicheskii polimorfizm i varianty tseruloplazmina pri gepatolentikuliarnoi degeneratsii.

The relationship between differences in the clinical polymorphism of hepatolenticular degeneration (Wilson's disease) and characteristics of CP (ceruloplasmin) structural changes were investigated. The comparative study of Wilson's disease patients revealed two forms of clinical development of this disease which differ from each other by the expression of the visceral symptoms preceding the establishment of the typical neurological picture. The peptide map analysis of tryptic hydrolysates of the CP from individual patients has demonstrated the altered peptide patterns in five cases. Clinical and genetic heterogeneity of Wilson's disease is discussed.

[The cytophotometric determination of the transferrin content of rat hepatocytes by using monoclonal antibodies]. / Tsitofotometricheskoe opredelenie soderzhaniia transferrina v gepatotsitakh krys s ispol'zovaniem monoklonal'nykh antitel.

Monoclonal antibodies specifically recognizing rat transferrin were produced. Optimal conditions of fixation and carrying out immunocytochemical reaction were determined. A combined method is proposed based on the indirect immunoperoxidase reaction and Feulgen procedure to be used for studying transferrin contents in hepatocytes with different ploidy. The transferrin content increased proportionally to the cell ploidy. It is concluded that the cytophotometric method may be used for a quantitative study of transferrin content in isolated rat hepatocytes.

[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.

[Spectrofluorometric analysis of protein fractions of blood serum of health donors and patients with kidney diseases]. / Spektrofluorimetricheskii analiz belkovykh fraktsii syvorotki krovi zdorovykh donorov i bol'nykh s zabolevaniiami pochek.

A comparison was made of ultra-violet fluorescence characteristics of albumin enriched protein fractions of serum blood of 35 glomerulonephritis (GN) patients, 30 uremic haemodialysis patients, and 40 donors. It has been found that fluorescence spectra of the albumin enriched fractions of serum from GN patients are blue shifted as compared with those of donors' serum preparations. At the same time fluorescence spectra of the albumin enriched fractions of donors' serum are similar, while those of GN patients vary significantly. The ratio of fluorescence intensities at 320 and 365 nm (A = I320/I365), characterizing the spectrum position is 1.27 +/- 0.05, for protein preparations of donors, while that for GN patients varies within the limits of 1.3-2.1. Fluorescence spectra of protein fractions from blood serum of uremic patients are red shifted (A = 0.77-1.29) in comparison with those of donors' blood. Chromatographic investigations show that protein preparations of GN patients' blood contain monomers and dimers of albumin, that can be divided according to their molecular masses or hydrophobic properties of the surface. Joint chromatographic and spectral analysis allowed to distinguish up to six albumin enriched fractions. A protein fraction with blue fluorescence spectrum was obtained by gel-filtration and ion exchange chromatography. A higher concentration of this fraction determines a high value of parameter A, that is typical for protein preparations of GN patients' blood. Amino acid sequence shows that one component of this fraction is beta-haptoglobin.

[Kinetics of inactivation of lectin: analysis of a method of tryptophan phosphorescence at room temperature]. / Kinetika inaktivatsii aktina: analiz metodom triptofanovoi fosforestsentsii pri komnatnoi temperature.

The internal dynamics of muscle actin during inactivation induced by guanidine hydrochloride (0.5-1.8 M) was studied by the method of room-temperature tryptophan phosphorescence (RTTP). It was shown that the essentially unfolded actin intermediate, which appears within the first minutes of incubation with guanidine hydrochloride, exhibits no RTTP, suggesting a high lability of its structure. Subsequent accumulation of associates of inactivated actin is accompanied by a significant increase in the intensity and decay time of RTTP, which is caused by the rigidity of the structure of inactivated actin. The kinetic dependencies of the intensity and lifetime of RTTP of actin during its inactivation depended on the concentration of the protein and guanidine hydrochloride.

[Interaction of lactoferrin with plasmid DNA studied by laser correlation spectroscopy]. / Kharakter vzaimodeistviia laktoferrina s plazmidnymi DNK po dannym lazernoi korreliatsionnoi spektroskopii.

The interactions of lactoferrin with plasmid DNAs substantially differing in the number of affine regions were studied. The dissociation constants for protein-nucleic acid complexes were determined. The possibility of standardizing the conditions for preparing multicomponent systems for gene-substituting therapy by laser correlation spectroscopy is discussed.

[Tryptophan phosphorescence of nascent and inactivated actin at the room temperature]. / Triptofanovaia fosforestsentsiia pri komnatnoi temperature nativnogo i inaktivirovannogo aktina.

Millisecond internal dynamics of native and inactivated actin from rabbit skeletal muscle was examined using room temperature phosphorescence. Inactivated actin was prepared by incubation of G-actin at 70 degrees C, by treatment with 4 M urea or 1.5 M guanidinium hydrochloride, renaturation from fully unfolded state or by Ca2+ ion removal. It was shown that inactivation of actin, irrespective of the denaturation procedure applied, leads to a sharp decrease of millisecond fluctuations of the protein structure. Restriction of the slow intramolecular mobility in inactivated actin can result from changes of the protein conformation and/or specific association of macromolecules.

[Analysis of species differences of radioactively labeled ceruloplasmin and albumin by means of peptide maps]. / Analiz vidovykh razlichii radioaktivno mechennykh tseruloplazmina i al'bumina s pomoshch'iu peptidnykh kart.

A procedure is developed for microanalysis of proteins, namely of human and rat ceruloplasmins as well as of their proteolytic fragments. The procedure involved electrophoretic purification of the peptides studied, labelling with 125I, tryptic hydrolysis and separation of the labelled peptides by cellulose thin-layer chromatography. Characteristics of the methods are described. Identification of individual polypeptide chains is reported. Specific localization of the labelled peptides on radioautogramms of the peptide maps was characterized. Analysis of human and rat ceruloplasmins, their fragments as well as human and rat blood serum albumins is presented as an example.

[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.

[Analysis of molecular microheterogeneity of plasma glycoproteins in a patient with sialidosis 1]. / Analiz molekuliarnoi mikrogeterogennosti nekotorykh glikoproteinov plazmy krovi bol'nogo sialidozom 1.

Using specific polyclonal monovalent antibodies, the molecular microheterogeneity of acute phase proteins: orosomucoid, alpha1-antitrypsin and ceruloplasmin, circulating in peripheral blood of a healthy donor and a patient with a hereditary deficiency of lysosomal neuraminidase (syalidosis I or the cherry stone syndrome), was analyzed by use of 2D electrophoresis. The specific distinctions due to a deficiency [caused by a deficiency] of lysosomal neuraminidase were revealed in the population of ceruloplasmin molecules, but not in the molecules of alpha1-antitrypsin and orosomucoid (alpha 1-acid glycoprotein). The molecular genetic bases of molecular microheterogeneity of some plasma glycoproteins and the possible use of natural models in studies of GP's functional role and the pathways of its transfer after internalization into non-hepatocytic (?) cells are discussed.