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Biomedical subjects

V V Demin

Publications and source records attributed to V V Demin.

At least 19 recordsLinked to original sources
PubMed2000

Atomic force and electron microscopy of high molecular weight circular DNA complexes with synthetic oligopeptide trivaline.

Intramolecular compact structures formed by high molecular weight circular superhelical DNA molecules due to interaction with synthetic oligopeptide trivaline (1) were studied by atomic force and electron microscopy. Three DNA preparations were used: plasmids pTbol, pRX10 and cosmid 27,877, with sizes 6,120 bp, 10,500 bp and 44,890 bp respectively. Plasmid pTbo1 and pRX10 preparations along with monomers contained significant amount of dimers and trimers. Main structures in all preparations observed were compact particles, which coincide in their appearance and compaction coefficient (3,5-3,7) with triple rings described earlier. The size and structure characteristics of triple rings and other compact particles on atomic force images in general coincide with those obtained by EM (2). AFM (3) images allow to get additional information about the ultrastructural organization and arrangement of DNA fibers within the compact structures. Along with triple rings in pTbol and pRX10-TVP complexes significant amount of compact structures were observed having the shape of two or three compact rings attached to each other by a region of compact fibre. Basing on the data of contour length measurements and the shape of the particles it was concluded that these structures were formed due to compaction of dimeric and trimeric circular DNA molecules. Structures consisting of several attached to each other triple rings were not found for pTbol, pRX10 monomers or cosmid preparations--TVP complexes where only single triple rings were observed. The conclusion is made that initiation of compact fibre formation within the circular molecules depends on the primary structure and for dimeric or trimeric circular molecules two or three compaction initiation points are present, located in each monomer unit within one circular DNA molecule. The nucleotide sequence dependent compaction mechanism providing independent compaction of portions of one circular molecule can be of interest for understanding of DNA compaction processes in vivo.

Cosmids↗
PubMed1998

High resolution mapping DNAs by R-loop atomic force microscopy.

R-loops formed by short RNA transcripts have been imaged by atomic force microscopy (AFM) at a constant force in the height mode. The technique was applied to mapping the human endogenous retrovirus K10 family (HERV-K10) long terminal repeats (LTR) within individual plasmids and cosmids. RNA probes specific for the U3 (384 nt) and U5 (375 nt) LTR regions separated by a span of 200 bp were used for R-loop formation with LTRs located within plasmid (3.8 kb) or cosmid ( approximately 40 kb) DNAs. R-loops stabilized by glyoxal treatment and adsorbed onto the mica surface in the presence of magnesium ions looked like looped out segments of RNA:DNA hybrids. The total yield of R-loops was usually approximately 95%. The RNA:DNA hybrids were found to be 12-15% shorter than the corresponding DNA:DNA duplex. The two regions of the LTR could be easily discerned in the AFM images as clearly separated loops. R-loop positions determined on cosmids by AFM were accurate to approximately 0.5% of the cosmid length. This technique might be easily adapted for mapping various sequences such as gene exons or regulatory regions and for detecting insertions, deletions and rearrangements that cause human genetic diseases.

Cosmids↗
PubMed1998

[Use of an antioxidant complex in the treatment of endotoxicosis in reconstructive surgery of lower limb arteries].

During a dynamic examination of 69 patients with obliterating atherosclerosis of the lower extremity vessels who were subjected to different reconstructive operations on the distal portion of the aorta and arteries the content of the dienic conjugate and malonic dialdehyde was studies both before the operation and during different stages of the latter, as well as at the postoperative period. It was found that the patients had higher content of these substances both before and after operations. A scheme is proposed for the complex antioxidant therapy which allows the degree of endotoxicosis resulting from the activation of lipid peroxidation to be decreased.

Adult↗
PubMed1996

Determination of the patterns of hexanucleotide distribution along DNA by electron microscopy.

Hexanucleotides containing modified bases (5-methylcytosine and 2-aminoadenine instead of cytosine and adenine) with increased capacities to bind complementary DNA sequences were used to map the distribution of their complementary sequences in a DNA target using electron microscopy. The method used hexamers to initiate DNA polymerase directed DNA synthesis at complementary sequences along a template. DNA synthesis was limited to about 200 residues by using a low concentration of deoxynucleotide precursors. During DNA synthesis a biotin ligand was incorporated to facilitate the subsequent binding of an electron-dense label (streptavidin-labeled colloidal gold particles) into newly synthesized DNA chains. The method can be implemented with commercially available products. The results demonstrate that the approach can be used to compare primary structural features of DNA fragments. The principles of the method can be adapted to a variety of single molecule detection methods such as electron, scanning tunneling, or atomic force microscopies.

Animals↗
PubMed1995

[Nonchemical methods of modifying substrates for studying biopolymers with atomic force microscopy].

Two simple procedures of DNA molecule fixation on mica for following imaging by the atomic force microscopy were developed. The distinctive features of the procedures are their simplicity, absence of chemical modification stages, and the possibility to obtain the images in air under relative high humidity. Comparison of the features of the images obtained indicated that the procedures developed were competitive with the procedures earlier suggested.

Aluminum Silicates↗
PubMed1993

Electron microscopy of two-dimensional crystals of mitochondrial ATP synthase.

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 microns in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at about 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a = 13.0 nm, b = 25.6 nm and gamma = 86 degrees. Two alternative models for the crystal structural organization were suggested, viz. with one or two protein molecules per unit cell.

Animals↗
PubMed1993

[Electron microscopic study of ATP synthetase from bovine heart mitochondria].

Two-dimensional crystals of the mitochondrial ATP synthase up to 0.4 microns in size were obtained from the detergent-lipid-protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier-filtering procedure at ca. 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a 13.0 nm, b 25.6 nm and gamma 86 degrees. In line with this conclusion, two alternative models for the crystal structural organization are plausible, viz., with one or two protein molecules per unit cell. The first model suggests an asymmetric incorporation of ATP synthase molecules into the lipid bilayer: extramembranous portions F1 are located on one side of the crystal membrane plane. According to the second model, the incorporation occurs on each side of the lipid bilayer, the unit cell containing the two oppositely oriented protein molecules. Based on the absence of another type of the projected crystal images (a rear view of the membrane), unique to the first model, preference is given to the second model.

Animals↗
PubMed1991

[Electron microscopy of alpha-latrotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus].

Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.

Crystallization↗
PubMed1991

Studies on the N-terminal sequences of lectins isolated from the seeds of Butea frondosa.

Two lectin fractions (FI and FII) were obtained from seeds of Butea frondosa by affinity chromatography on a sorbent of macroporous glass coupled to the disaccharide alpha-D-GalNAc-(1----3)-beta-D-Gal. Both of these fractions, although different in their sugar specificity, were found on SDS-PAGE to consist of two polypeptide chains of 33 kDa and 35 kDa. In the native state the subunits associated to form a 250 kDa complex, possibly comprising four molecules of the 33 kDa polypeptide and four molecules of the 35 kDa polypeptide. The presence of a faint 70 kDa band when the 250 kDa complex was subjected to SDS-PAGE may indicate the existence of a sequential mechanism of aggregation. N-terminal amino acid sequence analysis revealed extensive homology between these lectins and those of other Leguminosae.

Amino Acid Sequence↗
PubMed1990

Two-dimensional crystallization of reaction centers from Chloroflexus aurantiacus.

Two-dimensional crystals of photosynthetic reaction centers from Chloroflexus aurantiacus were obtained from protein-lipid-detergent micelles by detergent dialysis. The size of crystals was up to 2 microns. Some of them were multilayered crystals. However, other crystal forms were also observed. Preliminary image processing analysis showed that crystals of one crystal form referred to two-sided plane group p2 and had the following unit cell parameters: a = 17.6 nm, b = 18.0 nm, gamma = 84 degrees. The contour map of the crystal stain-excluding region was calculated by the Fourier-filtering procedure at about 2 nm resolution.

Bacterial Proteins↗
PubMed1990

Crystallization of membrane proteins: bovine rhodopsin.

The conditions for crystallization of the integral membrane protein bovine rhodopsin were determined. Crystals form over a wide range of protein concentration (0.4-15 mg ml-1) and pH (5.5-7.5) in detergent solutions of octyl-polyoxyethylene (o-POE) that contain inorganic salts as precipitants. Crystallization occurs in the detergent-rich phase under conditions of phase separation. This corroborates the results of experiments on the phase behavior of the detergent solution and on the distribution of rhodopsin between the salt-rich and detergent-rich phases formed at high salt concentrations. Crystals were needle shaped, and the best crystals were obtained by vapour diffusion, at room temperature, of a 2.5 mg ml-1 protein solution in a 1.5% (v/v) o-POE solution containing 1.5 M ammonium sulphate (pH 7.0) against unbuffered 2.85 M ammonium sulphate. The crystals thus produced had dimensions of approximately 70 microns x 70 microns x 1000 microns. These crystals were too small to allow X-ray diffraction studies of rhodopsin structure, but electron microscopic studies of negatively stained thin crystals allowed the definition of the projection belonging to the two-sided plane group p21, and showed unit cell parameters alpha (50 A), b (72 A), and gamma (90 degrees).

Animals↗
PubMed1986

[Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex].

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.

Animals↗
PubMed1985

Three-dimensional structure of (Na+ + K+)-ATPase revealed by electron microscopy of two-dimensional crystals.

Prolonged incubation of membrane fragments containing homogeneous (Na+ + K+)-ATPase with Mg2+, K+ and VO-3 at 4 degrees C resulted in formation of two-dimensional crystals of this enzyme with unit cell parameters: a = 66 A, b = 118 A, gamma = 108 degrees. The crystals correspond to the two-sided plane group p21. By combining tilted electron microscopic views of the crystals, a three-dimensional structure of (Na+ + K+)- ATPase was calculated at approximately 20 A resolution. The unit cell is formed by two (alpha beta)-promoters which are in contact in their central parts. The structure was compared with chemical modification and immunochemical data; the arrangement of intra- and extramembrane domains was proposed.

Animals↗
PubMed1976

[Energization of membrane vesicles from the cells of glycolyzing bacterium Streptococcus faecalis].

Membrane fractions were isolated from Streptococcus faecalis cells of a glycolyzing microorganism, devoid of the respiratory chain, using the methods of osmotic shock of the protoplasts, ultrasonic treatment of the cells and ultrasonic treatment of the protoplasts. All fractions possessed the ATPase activity, the highest activity being observed in the fraction isolated by ultrasonication of the protoplasts. All preparations were estimated with respect to the presence of vesicles, formed by the "inside-out" and "inside-in" membranes, using ATPase as a marker of the membrane orientation. In the membrane fractions obtained by ultrasonication of the protoplasts, the "inside-out" vesicles were prevalent. ATP-dependent energization of the membranes, sensitive to the action of dicyclohexylcarbodiimide and tetrachlorotrifluoromethyl benzimidazole, was demonstrated by measuring the transport of the lipophylic anion of phenyldicarbaundecaborane and aniline naphthalene sulfonate fluorescence.

Adenosine Triphosphatases↗