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Biomedical subjects

V N Morozov

Publications and source records attributed to V N Morozov.

At least 19 recordsLinked to original sources
PubMed2001

Immobilization of proteins in immunochemical microarrays fabricated by electrospray deposition.

Electrospray (ES) deposition has been applied to fabricate protein microarrays for immunochemical assay. Protein antigens were deposited as arrays of dry spots on a surface of aluminized plastic. Deposition was performed from water solutions containing a 10-fold (w/w of dry protein) excess of sucrose. Upon contact with humid air, the spots turn into microdroplets of sucrose/protein solution from which proteins were either adsorbed or covalently linked to clean or modified aluminum surfaces. It was found that covalent binding of antigens via aldehyde groups of oxidized branched dextran followed by reduction of the Schiff bonds gives the highest sensitivity and the lowest background in microarray-based ELISA, as compared to other tested methods of antigen immobilization. The minimum concentration of a primary mouse antibody detected in indirect ELISA with such antigen microarrays was approximately 0.3-1.0 ng/mL for ELF-97 or BCIP/NBT substrates of alkaline phosphatase.

Animals↗
PubMed1999

Electrospray deposition as a method for mass fabrication of mono- and multicomponent microarrays of biological and biologically active substances.

Electrospray of protein and DNA solutions is currently used to generate ions for mass spectrometric analysis of these molecules. Deposition of charged electrospray products on certain areas of a substrate under control of electrostatic forces is suggested here as a method for fabrication of multiple deposits of any size and form. For example, multiple dots of protein, DNA, or other organic substances can be deposited simultaneously through an array of holes in a dielectric mask covering any slightly conductive substrate (membrane, wet glass, semiconductor, etc.). If every new substance is deposited after a shift of the mask with respect to the substrate, a multicomponent matrix is created under each hole. It is demonstrated that dots as small as 2-6 microns can be fabricated by such an electrospray deposition (ESD). It is also demonstrated that the ES-deposited proteins and DNA retain their ability to specifically bind antibodies and matching DNA probes, respectively, enabling use of the ESD fabricated matrixes in Dot Immuno-Binding (DIB) and in DNA hybridization assays.

Alkaline Phosphatase↗
PubMed1997

Effect of nuclear protein HMG1 on in vitro slippage synthesis of the tandem repeat dTG x dCA.

Tandem repeats of simple doublet and triplet sequences occur with high frequency in the DNA of eucaryotes. Among the most frequent is the repeat of dTG, which has unusual structural properties. We show here that HMG1 (modeled by the second HMG box motif from HMG1 of the rat, HMGb) binds to complexes formed from annealing unequal lengths of dTG x dCA and inhibits the in vitro elongation of these complexes by the Klenow fragment of DNA polymerase I at 37 degrees C. At 46 degrees C, HMGb enhances the elongation. Polylysine inhibits elongation at both temperatures. These results show that the stability of this repeat in vivo can be influenced by the presence of basic proteins in general, and more selectively by the abundant nuclear protein HMG1.

Animals↗
PubMed1997

[State of cardiovascular and blood coagulation systems during prolonged exposure to extreme stimuli].

The studies covered complex effects of local shock vibration at extreme levels and ambient heat load with intensive impulse noise and general vibration in association with heavy physical work. The experiments on animals assessed influence of low temperatures on cardiovascular and hemocoagulation systems. Analysis of those clinical and experimental data proved that extreme and subextreme levels of long-acting factors induce disorders of hemostasis-depression of anticoagulation, that is similar to 1 phase of DIC syndrome. These results are important for diagnosis of premorbid and pathologic conditions.

Adult↗
PubMed1996

A magnetic micromethod to measure Young's modulus of protein crystals and other polymer materials.

A new micromethod has been developed to measure the elastic modulus of polymer materials. It is based on measurements of bending of a polymer sample in a periodic uneven magnetic field acting on a small permanent magnet attached to the sample free end. As compared to the other methods known, it combines simplicity of resonant methods with a possibility to perform measurements at different frequencies in liquids under normal and high pressures. The method is specifically designed to measure the temperature dependence of cross-linked protein crystals under different conditions.

Calibration↗
PubMed1996

Ionic conductivity, transference numbers, composition and mobility of ions in cross-linked lysozyme crystals.

Micromethods for measurements of electric conductivity, transference numbers and concentrations of inorganic ions within immobilized protein crystals have been developed and applied to study tetragonal lysozyme crystals cross-linked with glutaraldehyde. Donnan equilibria and mobilities of ions in this crystal were calculated using the data of these methods and the data of crystal pH titration. Taken together these results characterize the lysozyme crystal as an ion exchanger whose electrical properties and ion composition differ greatly from those of the external solution. Although anions transfer most of the current in the crystals, anion mobility is considerably lower than that of cations. Mobility of all ions in the crystal is considerably lower than in solution (3.5-50 times for cations and 120-330 times for anions) and depends on steric restrictions and charges of both ions and lysozyme molecules. Similar features in behavior of crystalline and biological channels are discussed.

Chemical Phenomena↗
PubMed1996

New polyacrylamide gel-based methods of sample preparation for optical microscopy: immobilization of DNA molecules for optical mapping.

New methods have been developed for rapid immobilization of biological macromolecules and other microscopic objects from aqueous solution at gel/gel, gel/solid and gel/solution interfaces using thin polyacrylamide gels covalently bound to glass surfaces. When quickly spread over a dry gel, an aqueous sample loses most of its water and low-molecular-weight solutes due to migration of these components into the gel. All optically observable objects thus become concentrated at the gel surface and may be easily located by light microscopy. Based on this, a procedure for binding DNA at a positively charged gel/solution interface was developed. A mild immobilization of the DNA molecules was obtained, allowing 'all in focus' observations of DNA digestion by restriction endonucleases with an apparent rate close to that in solution.

Acrylamides↗
PubMed1996

Stabilization of helical peptides by mixed spaced salt bridges.

Whether or not surface salt bridges have a strong stabilizing effect on the native structure in proteins remains uncertain. Previous studies of model peptides have shown that salt bridges spaced at i,i +4 along the chain are more stabilizing than those spaced at i,i +3, with a preference for the order acid-base rather than base-acid from N to C terminus. An analysis of the effect of spacing the ion pairs in short helical peptides is presented, in which acidic and basic side chains spaced two or three residues apart alternate along the chain. The mixed spacing proves to be stabilizing relative to pure spacings. A control peptide in which salt bridges were spaced uniformly three residues apart proved to form a beta-sheet structure rather than alpha-helix. This is due to formation of a silk-like apolar face consisting of alanine side chains; the mesoscopic structure formed by these sheets can be imaged by scanning microscopy.

Alanine↗
PubMed1996

[The blood coagulating activity in burns].

Data obtained in experiments in 80 rats and in clinical observations of 24 patients with burns of the II and IIIA degrees showed the activation of vasculo-thrombocytic and procoagulant links of the hemostasis system with a simultaneous depression of the anticoagulative blood system. The thermal trauma is followed by a dramatic decrease of the concentration of heparin, fibrinogen, decreased activity of antithrombin-III, fibrinolysis and plasminogen activators. There was a simultaneous growth of concentration of antiplasmins (alpha 2-macroglobulin and alpha 1-antitrypsin).

Adult↗
PubMed1995

Mechanical deformation enhances catalytic activity of crystalline carboxypeptidase A.

A new approach for investigating mechano-chemical interactions in enzymes is described. The catalytic activity of crystalline crosslinked enzymes subjected to uniaxial deformation has been measured. Extension of monoclinic P2(1) crystals of carboxypeptidase A along the [010] direction leads to a many-fold increase in catalytic esterase activity with no changes in the effective Michaelis constant. This increase is interpreted as due to liberation of conformational mobility associated with catalytic activity of the enzyme in the deformed crystal.

Carboxypeptidases↗
PubMed1993

Elasticity of globular proteins. The relation between mechanics, thermodynamics and mobility.

An analysis of elasticity of lysozyme and myoglobin crystals in terms of thermodynamics has revealed a direct relation between entropy and enthalpy of deformation and delta S* and delta H* terms in the standard free energy change in proteins, delta G(o), (K.P. Murphy, P.L. Privalov, S.J. Gill (1990) Science 247, 559-561), so that at any temperature (between the glass-transition and denaturation temperatures) free energy of deformation is proportional to the hydration independent part of delta G(o). Both energies are characterized with large enthalpy-entropy compensation and tend to zero at the same temperature, Tm = (delta H*/delta S*) = 353 +/- 20 K. Large positive entropy contribution to deformation energy causes large linear decrease in protein elasticity, and increase in thermal mobility of protein atoms with temperature. Being plotted in inverse coordinates, temperature dependence of the mean-square amplitudes, obtained in neutron and mossbauer experiments as well as in molecular dynamic simulations, gives the same 353 +/- 10 K for the temperature, where the amplitudes tend to infinity. Mechanism explaining large positive entropy contribution in deformation energy of native protein molecules presumably involves emergence of more room for motion of protein side-chain groups squeezed between alpha-helices and other rigid skeleton elements, when precise packing of atoms in native protein molecule is distorted as a result of deformation.

Crystallization↗
PubMed1993

A scanning tunnelling microscopy study of the formation and chemical activation of step defects on the basal plane of pyrolytic graphite.

Scanning tunnelling microscopy is used to monitor etching of the basal plane of highly orientated pyrolytic graphite by ozone, oxygen and nitric acid. These treatments are seen to produce numerous single and multilayer step defects. Subsequent modification of the graphite sheet edges flanking these cavities by cyanuric chloride, TiCl4 and other reagents is shown to activate the edges, thereby making them capable of covalently binding various molecules.

Acrylates↗
PubMed1993

New methods for depositing and imaging molecules in scanning tunneling microscopy.

Methods and apparatus are described to deposit and image molecules by scanning tunneling microscopy (STM) under an inert atmosphere. Three methods of applying molecules have been evaluated: equilibrium adsorption from the vapor phase, sublimation, and electrospraying. Using these methods, a variety of organic and biopolymer molecules have been deposited and imaged on graphite and on gold (111), grown epitaxially on mica. Compared with alternatives, such as the use of high vacuum apparatus or glove boxes, these procedures offer some important advantages: they are inexpensive, convenient, and more rapid. Mercaptoethanol, ethanolamine, ethanol, acetic acid, and water produce two-dimensional crystalline adlayers on gold substrates, when they are introduced into the scanning cell as vapors. These adlayers are assumed to involve hydrogen bonding of the molecules to an oxide of gold formed on the surface. Electrospraying protein solutions on gold surfaces yielded images of individual protein molecules with lateral dimensions close to those measured by X-ray analysis, and thicknesses of 0.6-1.3 nm. In the case of metallothionein, the known internal domain structure of the molecule was reproducibly observed. No detailed internal structure could be resolved in the other examples examined.

Acetates↗
PubMed1992

Mechanical detection of interaction of small specific ligands with proteins and DNA in cross-linked samples.

Cross-linked crystalline and amorphous films of different proteins and cross-linked DNA gels were found to change their mechanical properties when soaked in solutions of specific ligands at nearly physiological concentrations. This chemomechanical effect may be used to rapidly (within a few minutes) detect the ability of macromolecules to bind small (less than 1 kDa) ligand molecules, to measure concentrations of ligands (higher than 10 nM), and to estimate binding constants (lower than 10(7) M-1). Only 0.1-1 mg of protein or DNA is needed to prepare more than 10 samples sufficient for a large number of tests, provided binding is reversible. The method is recommended for rapid primary screening in search of new drugs, in biochemical studies, and as a basis for designing biosensors and other analytical instruments.

Animals↗
PubMed1992

A new micromethod for mechano-chemical research.

A new micromethod to study activity of enzymes in crystalline and amorphous solid samples subjected to tension is described. Both static (up to 200%) and dynamic (with an amplitude up to 50% and frequency of 10(-3)-50 Hz) deformation may be applied to the sample during the activity measurement. Strain-induced changes in activity of thin (3-10 microns) needle-like strips (0.2-0.5 microns long), cut out of the microtome sections of cross-linked crystals of pancreatic carboxypeptidase A is measured in a droplet of substrate solution (with volume of 10(-6)-10(-9) l) with a pH-microelectrode technique.

Carboxypeptidases↗
PubMed1991

Comparison of structures of dry and wet hen egg-white lysozyme molecule at 1.8 A resolution.

A high resolution structure of hen egg-white lysozyme containing 36 +/- 1 mol H2O per mol of protein has been obtained using triclinic (P1) crystals cross-linked with glutaraldehyde. Analysis of dehydration-induced structural changes has revealed displacement in relative position of domains and numerous small displacements in positions of individual atoms with r.m.s. deviation of main atoms 0.60 A, and that of all atoms 0.97 A. An increase in the average packing density of atoms in dry lysozyme by 4-6% seems to be the most probable reason for the loss of its activity and mobility.

Animals↗
PubMed1991

Survey of small molecule and ion binding to beta 2-microglobulin--possible relation to BEN.

Using a new method which permits rapid detection of ligand binding to cross linked protein films and crystals, we evaluated a spectrum of ligand binding to cross linked amorphous films of beta 2-microglobulin purified from urine of Balkan endemic nephropathy patients. Among more than 50 substances studied, including amino acids, fatty acids, sugars, nucleotides, nucleosides, and different inorganic ions, only polyvalent cations, such as Cu2+, Ca2+, Zn2+, Be2+ and La3+, were found to strongly bind to human beta 2-microglobulin with dissociation constants in the range 10(-6) to 10(-4) M. These cations can release beta 2-microglobulin from HLA complexes leading to an increased beta 2-microglobulin level in serum. These observations suggest a possible mechanism for the release of beta 2-microglobulin to make it available to exert toxic effects on the kidney as a potential pathogenesis of Balkan endemic nephropathy.

Balkan Nephropathy↗