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Biomedical subjects

V I Vorob'ev

Publications and source records attributed to V I Vorob'ev.

At least 19 recordsLinked to original sources
PubMed2006

The effect of manganese(II) on the structure of DNA/HMGB1/H1 complexes: electronic and vibrational circular dichroism studies.

The interactions were studied of DNA with the nonhistone chromatin protein HMGB1 and histone H1 in the presence of manganese(II) ions at different protein to DNA and manganese to DNA phosphate ratios by using absorption and optical activity spectroscopy in the electronic [ultraviolet (UV) and electronic circular dichroism ECD)] and vibrational [infrared (IR) and vibrational circular dichroism (VCD)] regions. In the presence of Mn2+, the protein-DNA interactions differ from those without the ions and cause prominent DNA compaction and formation of large intermolecular complexes. At the same time, the presence of HMGB1 and H1 also changed the mode of interaction of Mn2+ with DNA, which now takes place mostly in the major groove of DNA involving N7(G), whereas interactions between Mn2+ and DNA phosphate groups are weakened by histone molecules. Considerable interactions were also detected of Mn2+ ions with aspartic and glutamic amino acid residues of the proteins.

Animals↗
PubMed2004

The effect of manganese(II) on DNA structure: electronic and vibrational circular dichroism studies.

The interaction of DNA with Mn2+ was studied in absorbance and optical activity in the electronic and vibrational regions. Based on the data, several stages of the interaction were identified. Con formational transition towards the C-form of DNA was observed in solution at the molar ratio Mn2+/DNA-phosphates between 0.1 and 1.5. The exact ratio depended on the ionic strength and increased with increasing NaCl concentration. Although manganese interacted with the phosphates and bases of DNA at higher metal concentrations, it is unlikely that direct chelation occurred. A model for the interaction between manganese ions and DNA mediated by water is suggested destabilizing the double helix and partially breaking the hydrogen bonds between the base pairs. At high Mn2+ concentrations DNA aggregation was observed.

Circular Dichroism↗
PubMed2004

[Dissociation action of cationic peptides with hydrophobic radicals on functional coupling between serpentine type receptors and GTP-binding proteins].

The coupling of hormone-activated receptor and heterotrimeric G protein is an important step of the signal transduction through adenylyl cyclase signal system (ACS). The numerous literature data and own results show that G protein-interacting regions, that are localized in cytoplasmic loops of receptors, have considerable positive charge, can form amphiphilic alpha-helices and are tightly associated with the membrane. We studied the influence of model cationic peptides on both basal and stimulated by hormones and nonhormonal agents adenylyl cyclase (AC) activity and on GTP binding activity of heterotrimeric G proteins in skeletal muscles of rats and smooth muscles of mollusc Anodonta cygnea. Peptides with hydrophobic radicals of caprinoyl acid (C10): Lys(C10)-His-Glu-Lys-Lys-(C10)-His-Glu-Lys-Lys(C10)-His-Glu-Lys-Lys(C10)- His-Glu-Lys-Ala-amide (peptide I), Cys-Lys(C10)-X-Tyr-Lys-Ala-Lys7-Trp-Lys-amide (II), Cys-X-Trp-Lys-Lys(C10)-Lys2-Lys(C10)-Lys3-Lys(C10)-Tyr-Lys-Lys(C10)-Lys-Lys- amide (III), where X--epsilon-aminocaproyl acid residue, were synthesized by solid-phase methodology. IC50 values for inhibiting the influence of peptides on serotonin-(molluscs) and isoproterenol-stimulated (rats) AC activity were: for peptide I--56 and 70 mkM, for peptide II--32 and 47 mkM, for peptide III--22 and 28 mkM, respectively. At the same time the peptides weakly decreased AC activity stimulated by nonhormonal agents (NaF, Gpp[NH]p, forskolin). Peptides I--III stimulated basal activity of the enzyme in both investigated tissues. The maximum stimulating effects (28--52%) of the peptides were observed at their concentration 10 mkM. Peptides (10--100 mkM) increased Gpp[NH]p binding in plasma membranes of mollusc and rat muscles and strongly decreased the influence of the hormones on the binding. Based on the obtained data we supposed that cationic peptides with hydrophobic radicals mimic G protein-binding regions of the receptors and can be involved in the regulation of functional coupling between the receptors and G proteins.

Adenylyl Cyclases↗
PubMed2004

[Molecular mechanisms of action of dendrons, containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of the adenylyl cyclase signaling systems].

For the aims of studying molecular mechanisms of functioning of adenylyl cyclase signaling systems (ACS), we investigated the influence of synthetic polycationic peptides of the star-like structure (dendrons), containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of ACS components in smooth muscles of the mollusc Anodonta cygnea and in rat skeletal muscles. It has been shown that the following peptides (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx(= epsilon-aminohexanoic acid)-Cys(Acm), referred to as peptide I, (Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys(Acm) (peptide II), [(Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide III), and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide IV) inhibit in a dose-dependent manner the adenylyl cyclase (AC) activity stimulated by both nonhormanal agents (GppNHp and forskolin) and hormones, such as serotonin (mollusc) and isoproterenol (rat). Peptides III and IV (tetrameric dendrons) were most effective in comparison with peptides I and II (dimeric dendrons). The AC activity stimulated by hormones and forskolin was most sensitive to the action of dendrons. All dendrons stimulated GTP-binding activity of G-proteins: dimeric dendrons were most effective at 10(-5) M concentration, whereas tetrameric dendrons at 10(-6) M. In the presence of dendrons, the affinity of beta-antagonist [3H]-dihydroalprenolol to P-adrenergic receptor in rat muscle mem- branes was unchanged. At the same time, the affinity of beta-agonist isoproterenol to the receptor decreased, and no shift to the right was observed on the curve of isoproterenol-induced [3H]-dihydroalprenolol displacement in the presence of GTP. The obtained data show the disturbance of the coupling between the receptor and G-protein, which is the main reason of dendron inhibitory action on AC stimulation by hormones. Besides, these data demonstrated that hormones could disturb the functional activity of AC, i.e. a catalytic component of ACS.

Adenylyl Cyclases↗
PubMed2002

[Linker histones: conformational changes and the role in the structural organization of chromatin].

Histones, linker histones of the H1 family, their postsyntetic modifications, DNA-histone H1 interaction are reviewed. A question of protein change in spermatogenesis at the formation of inactive nucleus with high degree of DNA density is considered. Special attention was paid to sperm-specific histones of the H1 family of sperm cells. Their role in organization of high-order chromatin structure of sperm cells is discussed. Also, results of different studies on the structural organization of chromatin (nucleosomes, 30-nm fibers, chromatin loops and metaphase chromosomes) are discussed.

Animals↗
PubMed2000

[Interaction of superhelical DNA with the nonhistone protein HMG1].

The interaction between nonhistone chromosomal protein HMG1 and plasmid DNA was studied by optical and hydrodynamical methods. The recombinant protein HMG1 produced by yeast Pichia pastoris strain was used. We have shown that according to the CD spectra local conformational changes in DNA helix occur in the region of DNA-protein interaction. These changes are most significant at r < 3 (w/w). Both gel-shift assay and ultracentrifugation, as well as CD data, indicate that protein-protein interactions between HMG1 molecules play a major role in the formation of DNA-protein complexes. It is suggested that the protein C-terminus may affect HMG1-DNA binding not only by a direct interaction with DNA helix, but also by protein-protein interactions.

DNA, Superhelical↗
PubMed1998

Acid nuclear extracts as mediators of gene transfer and expression.

In an attempt to demonstrate transfection-active DNA packaging proteins in the cell nucleus, we prepared acid nuclear extracts with perchloric acid and subsequent protein fractions by stepwise acetone precipitation. The original extract and these fractions containing different compositions of nuclear proteins were used as DNA packaging agents. After the formation of complexes between these protein fractions and reporter genes, the addition of these complexes to the cells resulted in high transfection rates. Gel electrophoresis shows that the most active fractions contain histone H1 and HMG17. HMG1 exhibits a smaller activity. This result was confirmed by positive transfection experiments with commercial histone H1. Our results show that the transfection activity of acid nuclear protein fractions and histone H1 is dependent on the presence of calcium.

3T3 Cells↗
PubMed1998

[Chromatin compactification using a model system of DNA-protein complexes].

Conformational peculiarities of DNA complexes with histones of the H1 family have been studied by the method of circular dichroism (CD). The H1 histones were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius, starfish Aphelasterias japonica, and bivalve mollusc Chlamis islandicus and also from the rat thymus. It is shown that these sperm-specific histones do not compact DNA in low ionic strength solution. At physiologic conditions H1 from the sea urchin and starfish sperms compact DNA more intensively than other histones. The H1 from rat thymus has a minimum ability to compact DNA. This histone does not change the structure of DNA double helix. It was supposed that it could be associated with interactions of this histone with DNA in the major groove of its helix. At the same time sperm-specific H1 can interact with DNA not only in the major groove but also in the minor groove, and this induces changes in DNA structure. This DNA-protein interaction is specific for the sperm chromatin and may support the supercompact organization of the sperm chromatin.

Animals↗
PubMed1997

H1 and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer.

In this article we describe the chromatographic separation of acid nuclear protein fractions which have previously been shown to be active in DNA transfection experiments. By combining anionic and cationic ion exchangers, we were able to separate and identify some of the active proteins. In addition to HMG1, already known for its transfection activity, we have identified histone H1 and HMG17 as further transfection-active proteins. The highest transfection activity was associated with H1 and another nonidentified protein showing a somewhat higher electrophoretic mobility than H1. We have also found that the presence of CaCl2 in a low concentration in the cell culture medium is an important requirement for transfection.

Animals↗
PubMed1996

[An immunohistochemical study of the expression of transcription factor Oct3/4 in mouse spermatogenesis].

The expression of POU-domain transcription factor Oct3/4 in the testis of adult mice has been studied using indirect immunofluorescence with highly specific antibodies. The protein is shown to be expressed in germ cells of seminiferous epithelium in a stage specific manner. The protein synthesis is initiated in mid-pachytene spermatocytes, increases to reach its peak during meiotic division. The Oct3/4 level remains augmented in early spermatids, but gradually declines during their further developmental advancement. These findings imply that Oct3/4 may have a regulatory function providing for the control of meiosis and/or terminal differentiation of spermatogenic cells.

Animals↗
PubMed1995

[Conformational features of linker proteins of supercompact chromatin from marine invertebrate sperm].

A comparison of conformational potencies of linker histones of the H1 family has been performed by the curcular dichroism (CD) method. The histones were isolated from sera urchin (Strongylocentrotus intermedius), starfish (Aphelasterias japonica) and mollusc (Chlamis islandicus) sperm and rat thymus. The presence of an additional alpha-helical segment in the C-terminal part of the H1 histone from sea urchin sperm has been found to be a characteristic peculiarity of echinoderm sperm linker histones H1. The conformational properties of the H1 from starfish and sea urchin sperm are similar. The terminal domain of the mollusc sperm H1 has a more stable conformation of the left-handed helix in comparison with histones H1 of a different origin. It has been shown that the terminal fragments of the tested histones play an essential role in thermal stabilization of the secondary and ternary structures of the globular domains in these histones. The results obtained point to the difference in the mechanisms of supercondensed organization of sperm chromatin. The role of H1 histones in this process is discussed.

Animals↗
PubMed1994

Disturbances of nuclear condensation in human spermatozoa: search for mutations in the genes for protamine 1, protamine 2 and transition protein 1.

During spermiogenesis, the successive replacement of the somatic histones by basic proteins, the transition proteins and protamines, allows normal sperm nuclear condensation. It was suggested that disturbances in nuclear condensation may result in male infertility. Here we report the first molecular analysis of the structure of three genes which code for germ cell-specific nuclear proteins, namely protamine 1 (PRM1), protamine 2 (PRM2) and transition protein 1 (TNP1) in infertile men with disturbed sperm chromatin condensation. In 36 infertile men whose spermatozoa showed a positive reaction with aniline blue, which is an indication for the presence of histones in the nuclei, the complete nucleotide sequences of the coding regions and 5' and 3' untranslated regions of the three genes were evaluated. In addition, 10 infertile patients with oligoasthenoteratozoospermia were studied in the same way, as well as nine infertile patients whose spermatozoa showed a reduction of the protamine 2 content. We did not detect any mutation in the three genes in any of the patients. We assume that the disturbances in the sperm chromatin condensation of our patients, and those described in the literature, are not primarily due to mutations in the genes for PRM1, PRM2 and TNP1.

Base Sequence↗
PubMed1993

Human male infertility may be due to a decrease of the protamine P2 content in sperm chromatin.

Basic chromosomal proteins were extracted from the sperm of fertile and infertile human males. The relative proportions of protamine 1, 2, and 3 were determined by scanning microdensitometry following electrophoresis of total protamine in polyacrylamide gels. The findings were as follows: (1) The proportion of protamine P(2 + 3) in sperm obtained from infertile males was lower than that in fertile males. (2) Protamine P(2 + 3) in infertile human males showed reduced affinity to DNA. The possibility that some cases of human male infertility may be due to mutation within the protamine P2 gene is discussed.

Chromatin↗
PubMed1993

[DNA-dependent RNA-polymerase II from human placental nuclei].

The procedure for isolation and purification of RNA polymerase II from human placenta nuclei is described. The sensitivity of the enzyme to alpha-amanitin, bivalent cations, ionic strength and glycerol concentration in vitro has been studied. Eleven subunits of the RNA polymerase II molecule have been identified by gel electrophoresis. An optimal RNA polymerase II assay mixture has been developed.

Cell Nucleus↗
PubMed1993

[Features of structural organization of chromatin from various sources].

The conformational peculiarities of DNA and histones of chromatins of different origin have been studied using circular dichroism (CD). The chromatins were isolated from pigeon brain, rat thymus and liver, ascitic hepatoma 22A, C3HA mouse liver, pigeon erythrocytes and sea urchin sperm. The functional peculiarities of the chromatins were found to correlate with their compactness and the nucleosomal DNA repeat length. Analysis of chromatin CD spectra made it possible to define the degree of DNA compactness in oligonucleosomes and the secondary structure of their linker histones of the H1 family. It was found that in low ionic strength solutions the structures of chromatosomes are formed in erythrocyte and thymus chromatins, but not in sea urchin sperm chromatin. The size of the compact part of the DNA in the nucleosomes of transcriptionally active chromatins of brain and ascitic hepatoma 22A are less than the length of the DNA of the core particles under identical conditions. The secondary structure of the H1 histone from sea urchin sperm chromatin, unlike other linker histones of the H1 family, contains an additional alpha-helical segment in the C-terminal part. Analysis of structural changes of the both chromatin components during condensation of their oligonucleosomal chains with an increase in the ionic strength has been carried out.

Animals↗
PubMed1993

[Human male sterility can be caused by mutations in the protamine P2 gene].

The sperm of fertile and infertile human males was treated with 0.25 n. HCl or 1% CTAB. Relative proportions of various protamine fractions in the obtained extracts were determined by scanning microdensitometry following electrophoresis of the total protamine in polyacrilamide gels. The findings were as follow. 1. The share of protamine P(2 + 3) in the sperm obtained from infertile males was lower than that of fertile males. 2. Protamine P2 in infertile males has a reduced affinity to DNA. It is suggested that in some cases the human male infertility may be due to some mutations within the P2 gene.

Cell Count↗