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Biomedical subjects

R Tsanev

Publications and source records attributed to R Tsanev.

At least 19 recordsLinked to original sources
PubMed1999

Factors affecting the dissociation and aggregation of human interferon gamma.

The biologically active form of interferon gamma (IFN-gamma) is a dimer consisting of two identical non-covalently bound polypeptide chains. We have studied spectroscopically the dimer-monomer dissociation equilibrium of human recombinant IFN-gamma and have found that the monomers possess approximately 50% lower Trp quantum yield than the dimers [Boteva et al. Biochemistry 1996;35:14825]. In the present study we characterise the conformational properties of the two states--monomeric and dimeric, and analyse the effects of the salt composition of human blood plasma, physiological cations K+, Na+, Ca2+ and Mg2+ and mechanical stress on the dimer-monomer equilibrium. A medium with electrolyte composition of human blood plasma increases both the association and dissociation rate constants without shifting significantly the dimer-monomer equilibrium. The physiological cations shift the equilibrium towards dissociation of dimers into monomers by lowering the activation energy and the free energy of the process thus decreasing the stability of IFN-gamma. Mechanical stress caused by stirring of the protein solution reduces irreversibly the Trp fluorescence by 75-80% and decreases significantly the alpha-helical content and favours the aggregation.

Acrylamides↗
PubMed1996

Dissociation equilibrium of human recombinant interferon gamma.

The biologically active form of interferon gamma is a dimer composed of two noncovalently bound identical polypeptide chains of 17 kDa each. In this study, it was found that dissociation of the dimer into monomers significantly reduced the fluorescence quantum yield and the efficiency of the intermolecular Tyr to Trp radiationless energy transfer. The same process caused significant changes in the fluorescence decay and in the fluorescence anisotropy decay. The kinetic and thermodynamic parameters of the dimer-monomer equilibrium were determined by fluorescence measurements at different temperatures and by a theoretical mathematical model. Dissociation of the dimers into monomers was an endothermic process and was favored by concentrations of the protein lower than 1 microM and by increasing the temperature. It was accompanied by formation of aggregates, a slow and partially reversible process leading to inactivation of the interferon. It is suggested that certain monomeric conformers are competent for aggregation.

Biopolymers↗
PubMed1996

Effect of mechanical pressure on c-fos and on the mitotic activity of epidermal cells.

A dosed mechanical pressure of 12.5 kg/cm2 applied for 1 min on depilated mouse skin did not cause cellular death or visible alterations of the cellular ultrastructures. However, it had a strong effect on the mitotic cycle of the epidermal cells-stimulating the cells to enter the mitotic cycle and temporarily blocking the G1-->S transition. This effect was strictly limited to the pressed area of the skin. The proto-oncogene c-fos was induced within the first 2 min following application of the pressure. The level of c-fos mRNA showed two peaks during the next 24 h. The first slight peak was preceded by a rapid increase in the cAMP level in the pressed skin, the second-by a fall in the cAMP concentration. A model is suggested to explain the observed effects by reversible functional damage of the cellular membrane affecting the enzymes maintaining the steady state level of cAMP.

Animals↗
PubMed1994

Stably DNA-bound chromosomal proteins.

DNA accomplishes its biological function in a complex with nuclear proteins. A minor protein fraction has been found in chromatin which could not be dissociated from DNA by reagents abolishing non-covalent type of interactions. The controversy surrounding the nature of the protein moiety and the nature of the bond linking the two components on the one hand, and the fact pointing to its evolutionary conservatism and metabolic stability on the other, make it necessary to critically evaluate the data in view of the possible biological function for such proteins.

Animals↗
PubMed1994

DNA sequences tightly bound to proteins in mouse chromatin: identification of murine MER sequences.

The finding of stably (tightly) associated DNA-protein complexes in eukaryotic chromatin has provoked many hypotheses and speculations concerning their possible role. While the answer of this question is not envisaged yet, it is clear that elucidation of the nature of the individual components involved in such complexes is a necessary step in this direction. Here, the nature of several mouse DNA sequences in the vicinity of a putative stably attached protein is studied. Eight independently isolated clones containing such sequences were compared to known sequences in GenBank. Two clones were found to belong to different subfamilies of repetitive sequences, organized into a larger family--the L1md family. One clone harbors a sequence that is a member of the Alu-type family. Four of the cloned sequences are preset in low copy numbers, but the computer search found similar sequences in various genomic regions of different rodents. These facts, together with the finding that regions homologous to the above clones often flank other repetitive elements in the genome, suggest that the cloned sequences belong to new, not yet described families of repeats in the murine genome. It is possible that they correspond to the medium reiteration frequency sequences, MER-sequences, discovered recently in the human genome (Jurka, 1990; Kaplan and Duncan, 1990). Particularly intriguing is the homology found at the integration sites of polyoma virus in two transformed cell lines with two of these clones.

Animals↗
PubMed1993

Histone H1 deposition and histone-DNA interactions in replicating chromatin.

An immunochemical method for analyzing protein interactions with BrdUrd-substituted DNA was used to study binding of histones to nascent DNA in nuclei. The results indicate that in Ehrlich ascites tumor (EAT) cells, histone H1 deposits on newly replicated DNA simultaneously with or immediately after core histone deposition so that in chromatin replicated for 3 min, the stoichiometry of the histones is the same as in bulk chromatin. All histones, and especially histone H1, interact with nascent DNA more weakly than with bulk chromatin, although the efficiency of interaction via the globular domains seems to be the same for both types of chromatin.

Animals↗
PubMed1992

In vitro transcription through nucleosomes by T7 RNA polymerase.

The present work examines the fate of nucleosomes after in vitro transcription of a 1400 bp DNA template containing the mouse alpha-globin sequences and the promoter of T7 RNA polymerase. Naked and nucleosome-bearing templates (containing about four or seven histone H1-lacking particles per template) have been studied by sedimentation, gel electrophoresis, digestion with restriction nucleases and electron microscopy. Both naked and nucleosome-organized templates could be transcribed in vitro by the T7 polymerase. With all types of templates, both full length and shorter transcripts were obtained. The incomplete transcripts were represented by many distinct bands, pointing to the presence of multiple stops in the process of elongation. The electrophoretic pattern of the transcripts was identical in naked and in nucleosome-containing templates, showing that the stops depended on some particular DNA sequences and not on the presence of nucleosomes. The efficiency of transcription in the presence of nucleosomes was decreased owing to three different factors: (i) blocked initiation in a fraction of the templates which had their promoters occupied by a nucleosome; (ii) a decreased rate of elongation and (iii) a lag period of initiation. Sedimentation velocity, electrophoretic mobility and protection of four different restriction sites of the templates demonstrated that T7 polymerase transcribed through nucleosomes without their displacement.

Animals↗
PubMed1990

Interaction of histones H1 and H1(0) with superhelical and linear DNA.

By using direct competition experiments, the binding of histone H1AB (a mixture of H1A and H1B) and H1(0) to superhelical and linear DNA forms was studied. Mouse liver H1 isohistones and plasmid p alpha GD containing part of the 5' flanking and part of the coding sequence of the mouse alpha-globin gene in pUC18 were used as partners in the binding reaction. The competition experiments were performed by direct mixing of the histone with labelled supercoiled DNA (at 125 mM NaCl and at a histone/DNA ratio of 1.0) and addition to the mixture of increasing amounts of cold competitor DNA, either supercoiled or linear. The radioactivity of the complex formed was determined by filter binding. The results show that both histones H1 and H1(0) posses a strong binding preference for supercoiled DNA forms. Thus, histone H1(0) resembles the regular somatic set of histone H1 and not the other differentiation-specific histone H5 studied thus far.

Animals↗
PubMed1990

Protamine-histone replacement and DNA replication in the male mouse pronucleus.

The protamine to histone replacement in fertilized mouse eggs was studied by using antibodies to these proteins. Its course was followed with respect to DNA replication by autoradiography of 3H-thymidine-labeled fertilized eggs. It was found that protamines were replaced by histones before the onset of DNA replication.

Animals↗
PubMed1989

Histone variants in mouse centromeric chromatin.

Highly purified centromeric heterochromatin was isolated from mouse liver nuclei and the pattern of core histone variants was analyzed. In comparison with total chromatin, the centromeric heterochromatin of young animals was characterized by (1) enrichment in the replication-dependent variants H2A1, H2B2 and H3(2), (2) reduced amount of the minor variant H2Az and (3) absence of ubiquitinated molecules of H2A. This specific variant pattern changed upon ageing as a result of accumulation of replacement variants so that in adult animals both chromatin preparations exhibited similar pattern for H2A and H2B, while the difference in the profile of H3 variants was preserved.

Aging↗
PubMed1989

An evolutionarily conserved protein fraction stably linked to DNA.

Chromatins from four evolutionarily remote species (insect, fish, amphibian and bird) were isolated, high-salt-extracted and extensively deproteinized to remove noncovalently associated proteins. A protein fraction resisting the extraction procedures was found firmly linked to DNA in all four chromatins. Two-dimensional tryptic peptide mapping revealed a remarkable evolutionary conservativeness of this protein component, suggesting an indispensable function for it in the nucleus.

Animals↗
PubMed1988

Oxidation stability and fatty acid composition of selected storage and structural lipids: influence of different high fat diet compositions.

The oxidation stability of storage lipids from adipose tissue and of structural lipids from liver was compared to the coefficients of oxidizability of the pure fat used: lard (L); lard + sunflower oil 2:1 (LS); butter (B) and partially hydrogenated oil (H). The oxidation stability of the corresponding high-fat dietary regimens (50 energy-%) was also investigated. The experimental groups L, LS, B and H were compared to the control group (C) fed low-fat laboratory pellet-food. The coefficients of oxidizability were calculated from the fatty-acid composition of the used pure fats. The oxidation stability was performed in condition of accelerated oxidation under kinetic regimen, assaying the peroxide concentration in appropriate time intervals. The coefficients of oxidizability of dietary fats and storage lipids were very similar. This is explained by the fact that the fatty-acid composition of storage lipids reflected that of the corresponding high-fat diets. The oxidation stabilities in storage lipids were markedly lower than these in the respective dietary regimens. The highest oxidation stability in lipids from adipose tissue was found in group B, and the lowest in group LS. Contrarywise, the oxidation stability in liver lipid showed the following declining sequence: C greater than H greater than L greater than LS greater than B. The discrepancies in oxidation stability of the various specimens (pure fats, dietary fats, storage and structural lipids) may be explicated by an intervention of factors with pro- and anti-oxidative action. The large deviations in fatty acid composition in the examined tissues in comparison to the composition of the respective high-fat diets may also play an important role in this respect. These parallel studies on oxidation stability at different levels could improve our possibilities for evaluation of the stability and biological value of fats.

Adipose Tissue↗
PubMed1988

Immunofluorescent localization of histone H10 in the nuclei of proliferating and differentiating Friend cells.

By using affinity-purified antibodies to H10 and to H1AB the localization of these histones was studied by indirect immunofluorescence in the nuclei of proliferating (EAT and uninduced Friend cells) and of differentiating (induced Friend cells) cell populations. While with H1AB antibodies a bright fluorescence all over the chromatin was obtained, the localization of H10 varied depending on the state of the cell population. In the proliferating EAT cells it was localized strictly in the nucleoli. The Friend cell population revealed a heterogeneous picture with two types of H10 localization-nucleolar predominating in uninduced cell populations and peripheral predominating in induced cells. A comparison with literature data suggests that H10 seems to be associated with chromatin regions containing active genes.

Animals↗
PubMed1988

Metabolic behaviour of a stable DNA-protein complex.

1. A stable DNA-protein complex resisting all treatments dissociating noncovalently associated proteins was isolated from mouse erythroleukemia cells. 2. Two-dimensional tryptic peptide mapping of this DNA-linked protein component revealed a remarkable similarity to the maps of the corresponding proteins from other mammalian chromatins. 3. Labelling experiments showed that the protein component was metabolically stable.

Animals↗
PubMed1987

Stable DNA-protein complexes in eukaryotic chromatin.

Demembranized sperm and somatic nuclei of mammalian origin were extracted with high salt/urea/2-mercaptoethanol, treated with detergents and purified in CsCl density gradients to isolate DNA. Under these conditions a protein component still remained bound to DNA. This stable DNA-protein complex could be reduced to an oligodeoxynucleotide-peptide complex by extensive sequential digestions with DNase I and Pronase E. Chemical and enzymatic treatments of this complex indicated the presence of a phosphoester bond between DNA and a hydroxyamino acid. Two-dimensional tryptic peptide mapping revealed a remarkable similarity among the covalently linked protein components in all types of chromatin studied. These maps differed from the maps of mammalian topoisomerases I and II.

Animals↗
PubMed1987

Segregation of nucleosomes in replicated mouse alpha-globin gene.

DNA of mouse erythroleukemia cells grown in vitro was labeled with bromodeoxyuridine during cycloheximide-inhibited protein synthesis. Isolated nuclei were digested with micrococcal nuclease to obtain monosomes and monosomal dsDNA. The protection of the heavy and of the light strands of the newly replicated DNA was studied by dot hybridization with the coding and with its complementary noncoding strand of the alpha-globin gene. The results show that both sides of the replication fork contain protected sequences of the gene, thus supporting a bilateral (dispersive) mode of nucleosome segregation during DNA replication.

Animals↗
PubMed1987

Immunological evidence for the existence of H1-like histone in yeast.

In view of the controversies about the existence of histone H1 in yeast we have reinvestigated the problem by studying yeast proteins extracted with perchloric acid and salt. Perchloric-acid-extracted proteins from whole cells contain only two fractions which comigrate with 'authentic' yeast high-mobility-group proteins (HMG) in both SDS and acid urea gels. These extracts show a considerable cross-reaction with anti-(calf thymus HMG) antiserum and do not react with antiserum to mouse liver H1. The isolation of 'authentic' yeast HMG by the standard salt/trichloroacetic acid procedure gives two types of preparations containing different numbers of protein bands. The poorer preparation reacts only with the anti-HMG antiserum whereas the richer preparation also gives considerable cross-reaction with the anti-H1 antiserum. Immunoblotting analysis performed on the salt-extracted proteins reveals the presence of three protein bands giving positive immunoreaction with the anti-H1 antiserum. The immunoreactive bands have electrophoretic mobilities close to that of the marker calf thymus H1 and similar to the mobilities of the presumptive yeast H1 fractions found by other authors.

Animals↗