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Biomedical subjects

I A Kashparov

Publications and source records attributed to I A Kashparov.

At least 19 recordsLinked to original sources
PubMed2006

Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

Calcium↗
PubMed2004

Selective accumulation of monoclonal antibodies against neurospecific enolase in brain tissue of rats with middle cerebral artery occlusion.

Preparations of I(125)-labeled monoclonal antibodies against neurospecific enolase and mouse plasma IgG1 were injected intravenously to rats immediately after unilateral occlusion of the middle cerebral artery. Radioactivity of I(125)-labeled monoclonal antibodies against neurospecific enolase in the brain tissue progressively increased, reached a maximum by the 48th hour, and remained practically unchanged after 72 h. At the same time radioactivity of labeled IgG1 in the brain tissue and radioactivity of both preparations in the blood, liver, spleen, kidneys, heart, and lungs decreased over 72 h. Selective accumulation of I(125)-labeled monoclonal antibodies against neurospecific enolase was less significant in the brain tissue of the contralateral hemisphere and cerebellum not exposed to ischemia.

Animals↗
PubMed2004

Increased level of beta-amyloid in the brain of bulbectomized mice.

Six weeks after bilateral olfactory bulbectomy, a peptide with molecular weight of 4 kD was revealed in extracts of the neocortex and hippocampus from mice. Using monoclonal antibodies 4G8, this peptide was identified as beta-amyloid. Its level was significantly higher in the bulbectomized animals than in sham-operated mice. The bulbectomized mice displayed sharp impairment in spatial memory when tested in the Morris water maze. The results suggest that bulbectomy initiates in the brain a pathological process similar to human Alzheimer's disease in location, biochemistry, and behavioral manifestations.

Alzheimer Disease↗
PubMed2003

Antimicrobial peptides containing arginine.

Tetradecapeptides (RLARLAR)2, D-(RLARLAR)2, (RLARLAA)2, and (RLGRLGR)2 were synthesized by a solid phase method using Fmoc-amino acids. The antibacterial activity of the synthesized peptides was studied against Escherichia coli cells. The minimum inhibitory concentration (MIC) was, correspondingly, 3, 1, 3, and 12 micro M, which is comparable with MIC of such natural antimicrobial peptides as temporin, magainin, and dermaseptin. It was found that all of the synthesized peptides have no effect on human erythrocytes and rat thymocytes. The peptides form alpha-helices in 30% trifluoroethanol and in 2.5 mM SDS, which have amphipathic structure.

Amino Acid Sequence↗
PubMed1998

[Free amino acids in the synthesis of peptides. Effect of inorganic compounds on the solubility of amino acids in aprotic solvents].

In order to increase the solubility of free amino acids so that they could be used in peptide synthesis, a system consisting of dimethylformamide, an inorganic additive, and pyridine was suggested. Compounds BF3, AlCl3, SnCl2, SiCl4, ZnCl2, SbCl3, CaCl2, BaI2, CdI2, and NaI can serve as the additives. Acylation of amino acids in such a solution (pH 5.2-5.3) with pentafluorophenyl esters of N alpha-protected amino acids gave N alpha-protected di- and tripeptides in a yield of more than 90%.

Acylation↗
PubMed1997

Ligands regulate GroEL thermostability.

Escherichia coli heat-shock proteins GroEL and GroES stimulate (in an ATP-dependent manner) the folding of various proteins. In this study scanning microcalorimetry was applied to investigate GroEL thermostability in the presence of its ligands. Mg2+ and K+ ions stabilize while ADP destabilizes the GroEL molecule against the action of temperature. Furthermore, ADP essentially increases the number of binding sites for the hydrophobic probe (ANS) and the number of GroEL SH-groups accessible to Ellman's reagent as well as the accessibility of the protein to the action of trypsin. The interaction of GroEL with GroES in the presence of Mg2+-ADP eliminates the destabilizing effect of ADP on the GroEL molecule against the action of temperature and Ellman's reagent but does not change its hydrophobicity and accessibility to trypsin.

Adenosine Diphosphate↗
PubMed1995

Transport of hydrophobized fragments of antibodies through the blood-brain barrier.

Hydrophobized and non-hydrophobized Fab fragments of human antibodies against gliofibrillar acid protein (GFAP) and brain specific alpha 2-glycoprotein (alpha 2GP) were used to study their penetration through the blood-brain barrier (BBB). These Fab fragments were modified by stearoyl chloride in reversed micelles of aerosol OT in octane (one or two fatty acid residues attached to protein molecule). Modified and non-modified 125I-labelled Fab fragments were intracardially administered to rats. The amount of label accumulated in brain was 55% higher than the total amount in all other organs. In contrast, non-hydrophobized Fab fragments did not penetrate through the BBB. We assume that the artificial hydrophobization of Fab fragments can increase their capability to penetrate through the cell membranes and, in particular, the BBB.

Animals↗
PubMed1995

[Preparative isolation of proteins from 30S ribosomal subparticles from Thermus thermophilus under nondenaturing conditions].

A procedure for isolation in preparative amounts of 15 individual proteins from ribosomal 30S subparticles of Thermus thermophilus under non-denaturing conditions, has been developed. The amino acid composition and molecular masses of the proteins have been determined and the UV absorption spectra and extinction coefficients measured. A homology of 13 proteins to corresponding ribosomal proteins of E. coli has been established.

Amino Acid Sequence↗
PubMed1994

Proteins of the Thermus thermophilus ribosome. Purification of proteins from the large ribosomal subunit.

Special procedures have been developed to isolate and purify 26 of the 30 individual proteins of the large ribosomal subunit from Thermus thermophilus. Sixteen of them have been purified under non-denaturing conditions to be used for crystallization and further structural studies. These proteins have been characterized by their amino acid content, molecular mass, UV-spectrum and extinction coefficient. An additional 10 proteins have been purified by reverse phase chromatography. Thirteen proteins have been identified by homological E coli proteins.

Amino Acid Sequence↗
PubMed1994

Synthesis and properties of the peptide corresponding to the mutant form of the leucine zipper of the transcriptional activator GCN4 from yeast.

A 33 membered polypeptide corresponding to the leucine zipper region of the yeast transcriptional activator GCN4 was synthesized by solid phase chemical synthesis and characterized. Asparagine in the hydrophobic core of the molecule is replaced by valine in the synthetic variant. The correctness of amino acid sequence of the preparation is corroborated by direct sequencing. High-speed equilibrium ultracentrifugation, ultraviolet circular dichroism spectroscopy and scanning microcalorimetry have been employed to demonstrate that in solution the peptide forms a highly stable triple-stranded alpha-helical coiled coil. The stability of the mutant form is 40 degrees C higher than the dimeric form of natural peptide under similar conditions. It was proposed that location of some polar groups in the 'a' and 'd' positions of natural two-stranded coiled coils may be regarded as protection against alternative triple- and multistranded conformations.

Amino Acid Sequence↗
PubMed1989

[Status of the blood-brain barrier in newborn infants of various gestational ages in the normal state and in pathology].

The blood-brain barrier (BBB) was studied in 76 neonates of different gestation age in health and disease by means of detecting specific alpha-1-globulin in blood serum with the aid of ELISA. It has been established that by week 28 of the intrauterine development the process of the structural and functional establishment of the BBB had been over as evidenced by the lack of specific alpha-1-globulin in umbilical blood of the neonates of the given gestation age. Severe chronic intrauterine hypoxia combined with acute hypoxia resulted in brain damage and BBB opening for antigen penetration in the direction brain-blood. The measurement of the concentration of alpha-1-globulin in the course of observing the neonates made it possible to predict the degree of the CNS damage.

Alpha-Globulins↗
PubMed1981

Interaction of the N-terminal and C-terminal domains of elongation factor G on formation of complexes with guanyl nucleotides.

Polarized fluorescence studies of interaction between guanyl nucleotides (GTP and GDP) and elongation factor G and its N-terminal tryptic fragment T*/s, carrying a fluorescent group (aminorhodamine B) at the exposed cysteine residue, has shown that binding on nucleotides by an intact EF-G molecule at neutral pH essentially affects the mobility of the fluorescent group. GTP binding changes its relaxation properties to a greater extent than GDP binding. At the same time it was demonstrated that the spectrum of relaxation time of the fluorescent group practically does not change on binding of nucleotides by the N-terminal fragment T*/2 (in the absence of the C-terminal domain) or in the case when the three-dimensional structure of the intact EF-G molecule is destabilized (pH 10). Comparison of the relaxation properties of EF-G and its N-terminal fragment T*/2, carrying a fluorescent group at the exposed cysteine residue, at pH 7.5 and 10, indicates that the C-terminal domain is involved in the formation of the close environment of the exposed cysteine residue located in the N-terminal part of EF-G. A conclusion is drawn on the nucleotide-induced influence of the C-terminal domain on a change of the exposed cysteine residue environment on guanyl nucleotide binding with EF-G at neutral pH and a hypothetical model of the EF-G molecule is proposed.

Fluorescence Polarization↗
PubMed1981

[Properties and role of tryptophan residues in the polypeptide chain of elongation factor G from E. coli].

Using chemical modification and spectrofluorimetry, it was shown that two tryptophane residues of the elongation factor G (EF-G) in positions 51 and 71 from the N-terminus are located on the surface of the EF-G molecule. The tryptophane residue in position 71 is effectively shielded against modification at binding of EF-G guanyl nucleotides. Modification of these tryptophane residues does not result in a loss of the nucleotide-binding activity but completely inhibits EF-G binding to the ribosome. Polarized fluorescence study showed that the relaxation properties of these exposed tryptophane residues essentially depend both on the presence of the C-terminal domain and on binding of nucleotides in the nucleotide-binding site located in the N-terminal part of EF-G. It was assumed that the C-terminal domain, the nucleotide-binding site and the site responsible for the EF-G binding to the ribosome are brought together in the three-dimensional structure of the elongation factor G.

Escherichia coli↗
PubMed1980

Tyrosine residues in the C-terminal domain of the elongation factor G are essential for its interaction with the ribosome.

Chemical modification of the elongation factor G (EF-G) with tetranitromethane and iodine has been studied. It has been shown by spectrophotometric titration that EF-G contains two exposed tyrosine residues, one of which has an unusually low pK value for a phenol hydroxyl group at pH 8.5. Modification of one tyrosine residue with either tetranitromethane or iodine results in a 70--80% loss of EF-G activity in all ribosome-dependent reactions. Modification of three or four residues inhibits 90--100% of activity. Binding of EF-G with the 70-S ribosome and 50-S subunit is equally effective for protection of tyrosine residues against modification. The rate of EF-G modification with tetranitromethane is considerably higher in the presence of guanyl nucleotides than for free EF-G. The modified residues are located in the C-terminal domain of EF-G and are presumably contained in one of the sites of EF-G interaction with the ribosome.

Escherichia coli↗
PubMedDate not supplied

[Catechol siderophore, produced by thermoresistent strain of Bacillus licheniformis VK21].

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe3+, in response to addition of manganese(II) salts. The growth of the producer on a minimum medium containing MnSO4 under the conditions of iron deficiency is accompanied by the accumulation of a catechol product, the content of which reaches a maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl3, the amount of the catechol product in the medium considerably decreases. The siderophore, called SVK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to amino acid analysis and NMR spectrometry, the metabolite SVK21 is 2,3-dihydroxybenzoyl-glycyl-threonine. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

Bacillus↗
PubMedDate not supplied

PEGylated immunoliposomes directed against brain astrocytes.

Polyethylene glycol (PEG)ylated (stealth) immunoliposomes directed against human gliofibrillary acidic protein (GFAP) were prepared by coupling the thiolated monoclonal anti-GFAP antibodies with a maleimide derivative of phosphatidyl ethanolamine of the liposomal membrane. Experiments with cell cultures demonstrated specific and competitive binding of these immunoliposomes to embryonic rat brain astrocytes. Administered intravenously into rats, the immunoliposomes displayed typical kinetics with elimination half-lives of 8-15 hr. Being incapable of penetrating the unimpaired blood-brain barrier (BBB), these immunoliposomes, nevertheless, may be useful in delivering drugs to glial brain tumors (which continue to express GFAP) or to other pathological loci in the brain with a partially disintegrated BBB.

Animals↗
PubMedDate not supplied

[Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH].

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.

Apoproteins↗