Biomedical subjects
Publications and source records attributed to I A Iamskov.
Glycosylation procedures and their application for the elucidation of glycoprotein structure and structure-function relations, as well as for the development of analytical systems for clinical practice are reviewed. For some common cases found in research practice, the choice of optimal deglycosylation methods is discussed. Current views on the primary structure of glycoproteins are described briefly.
The relative hydrophobicity of ten purified lectins preparations (SBA, LCA, PNA, PSA, STA, WGA, Con A, PHA-P, RCA-I and HPA) was determined using the aqueous two-phase polymeric system Ficoll 400-dextran 70 (13.53% - 11.59%, w/w in H2O, respectively). The linear correlation was found between the relative hydrophobicity of the lectin preparations and their hemagglutinating activity towards native rabbit erythrocytes (r2 = 0.925). Con A specific carbohydrate ligands added to the system decreased the relative hydrophobicity of lectin. Using cross partition, isoelectric points for the lectins were determined.
Penicillinamidohydrolase was immobilized on heteroporous polystyrene supports containing groups of ethylenediamine, diethylenetriamine and triethylenetetramine, using Co(II), Ni(II), Cu(II), Fe(III), Co(III) ions. The effects of substrate concentrations and pH on the activity of the immobilized enzyme were investigated and the kinetic parameters of hydrolysis were determined, using N-phenacetylphenylglycine as substrate. The enzyme immobilization resulted in a slight decrease of Km(app), while the values of V decreased by a factor of two. Preparations possessing high thermal stability were obtained by oxidation of Co(II) to Co(III) in the enzyme sorption complex. High stability was also found for the Fe(III)-containing preparations.
The kinetics of N-acetyl-L- and N-acetyl-D,L-methionine hydrolysis by aminoacylase from Asp. oryzae were studied. The maximal rate of the reaction was found to be about 2000 mu moles of L-methionine per mg of protein per hour; Km was equal to about 1.10(-1) M for both the racemic and optically active substrates. In the presence of Co(II) ions (the molar ratio of N-acetyl-L-methionine/Co(II) was 100:1) the reaction velocity was increased. The values of approximately 4500 and approximately 3000 mu moles per mg per hour were-attained with L-enantiomer and racemic substrates, respectively. The Km values were decreased down to 2.10(-2) M and 1.4.10(-2) M, respectively.
Two groups of proteins were isolated from the retina and pigment epithelium of eight-day-old chick embryos. Experiments with suspension cultures of retinal cells demonstrated that only the retinal extracts and the fraction of its acidic proteins can stimulate cell aggregation in vitro. Analysis by the method of high-performance liquid chromatography showed that fractions of acidic and basic retinal proteins, which markedly differ in their electric charge and biological activity, have similar composition. To study the effect of these proteins on the morphological and functional state of pigment epithelium in vitro, a new experimental model is proposed, with the posterior segment of the newt (Pleurodeles waltl) eye used as a test tissue. The fraction of basic proteins isolated from the chick embryonic pigment epithelium stabilized cell differentiation in the newt pigment epithelium. The analyzed proteins proved to be biologically active at extremely low doses, corresponding to 10(-12) M solutions.
Amino acid composition, structure, and physicochemical properties of a low-molecular-weight glycoprotein from cattle blood serum (SGP) were studied. The content of carbohydrates (represented by mannose-rich oligosaccharides) amounted to 45-50 wt %. The value of specific partial heat of SGP, measured by differential scanning calorimetry (DSC), equaled 1.8 J/g.K, which is characteristic of unfolded proteins. Circular dichroic (CD) spectra of SGP led us to conclude that it is not highly structured and that it occurs in the shape of a statistical globule. The protein was deglycated using anhydrous trifluoromethane sulfonate (TFMS), after which its amino acid composition and the sequence of a fragment were determined. The results indicate that SGP is a protein not studied previously.
A lectin was isolated from the venom of scorpion Buthus occitanus sp. by means of Sephadex G-50 gel filtration and CM-cellulose ion exchange chromatography. The homogeneous lectin preparation consisted of homodimeric molecules with a subunit Mr of 9.3 kDa. Glycine, alanine, and serine dominated in the lectin amino acid composition. The lectin was a glycoprotein containing 20% carbohydrates (predominantly mannose and glucose). Trypsin-treated murine erythrocytes agglutinated at the lectin concentration of 32 micrograms/ml. Hemagglutination was inhibited by carbohydrates (L-fucose > D-glucose > L-rhamnose > D-xylose). The lectin revealed no phospholipase or hyaluronidase, nor toxic activity.
The possible application of polyelectrolyte complexes (PEC) of chitosan and copolymers of maleic acid with N-vinylpyrrolidone, styrene, and ethylene and/or chitin cross-linked sorbents (CCLS) synthesized on the basis of PEC for sorption of wheat germ agglutinin (WGA) was studied. The synthesis of spherically granulated sorbents was shown. Compared to unmodified chitosan, there was a significant increase in sorption of WGA by the PEC: PEC, 2.5-fold, and CCLS, from 3.5-fold to 7-fold.
The specificity of perch (Perca fluviatilis) roe fucolectin was studied using the protein dot blot technique, followed by detection with colloidal gold-labeled neoglycoproteins bearing human milk polysaccharides. The strongest binding was noted with the H type 1 pentasaccharide lacto-N-fucopentaose (LNFP I, Fuc alpha 1-2 Gal beta 1-3 GlcNAc beta 1-3 Gal beta 1-4Glc); the interaction with the H type 6 trisaccharide 2'-fucosyllactose (2-FL, Fuc alpha 1-2 Gal beta 1-4 Glc) was weaker. Binding of the perch lectin to the Lewis antigens (associated with tumors and embryonic tissues) was also studied. It was found that the lectin weakly interacted with the hexasaccharide lacto-N-difucohexaose I, Fuc alpha 1-2 Gal beta 1-3[Fuc alpha 1-4]GlcNac beta 1-3 Gal beta 1-4 Glc), but not with Lea, Lec, Lex antigens. Thus, perch roe lectin exhibited pronounced differences in carbohydrate specificity from other fucolectins--a feature that may be used in structural studies and isolation of fucose-containing glycoconjugates.
The complete amino acid sequence of an important toxin (toxin 14) from the venom of a Vietnamese scorpion (Buthus occitanus sp.) has been determined, which includes 35 amino acid residues and three disulfide bridges (molecular weight, 3843 Da). The comparison of the sequence with sequences of short scorpion toxins led us to conclude that toxin 14 belongs to a novel group of toxins affecting the excitability of myelinated nerves.
In our work the new proteins likely belonged to the microenvironment of pigmented epithelium cells and retinal neurons in mammalian eye were studied. We attempted to understand the role of these proteins in the maintenance of normal morphological and functional state of these eye tissues. Earlier for the first time we identified the adhesion molecules with physico-chemical and biological properties much different from other known cell adhesion molecules of bovine eye. Probably, they represent one family of low molecular weigh, highly glicosylated proteins, that express biological activity in extremely low doses--10(-10) mg/ml. The homogeneity of studying proteins is confirmed by HPLC and SDS-electrophoresis in PAAG. It is shown also that these proteins are N-glycosylated, because they contain mannose and N-acetilglucosamine residues. They demonstrate as well a high calcium-binding activity, with Kd corresponded to 10(-4)-10(-6) mg/ml. For a study of the biological effect of these glycoproteins in extremely low doses, a new experimental model was proposed and developed. It was the cultivation in vitro of the posterior part of the eye obtained from the newt Pleurodeles waltl. In short-time culture system it was demonstrated that the studied glycoproteins could stabilize pigment epithelium cell differentiation and cellular interactions in the neural retina in vitro. In addition, glycoproteins, obtained from the pigmented epithelium of bovine eye could decrease the rate of bipolar cell apoptosis in the neural retina. Therefore, the novel adhesion glycoproteins, expressing their biological activity in extremely low doses, pretend to be the regulatory molecules with vivid gomeostatic effects necessary for the delicate adjustment of cell behavior action and function in sensory tissues.
Searching and study on regulatory proteins, which can keep under control the scope of important processes as like as cell adhesion, proliferation, differentiation and morphogenesis, is an actual aim of the current biochemistry. Recently we have identified S-100 proteins in plants of following species: plantain (Plantago major L.), aloe (Aloe arborescens L.), and bilberry (Vaccinum myrtillus L.). Extraction and purification of S-100 proteins gotten from these plants were performed by the method we developed earlier for adhesion proteins of animal tissues. Homogeneity of the studied plant proteins was evaluated and confirmed by HPLC and SDS-electrophoresis in PAAG. Both, plant and animal proteins have appeared to be biologically active at extremely low doses. The tests were performed by adhesiometrical method in short-term tissue culture of mouse's liver in vitro. As a result it was established that the plant proteins insert a membranotropic effect being added in extremely low doses, corresponding to 10(-10)-10(-13) mg/ml. Keeping in mind that the plantain is well known remedy for wound protection and healing, in several experiments we studied the biological effect of plant S-100 proteins on animal cells. It was found that S-100 proteins obtained from plantain influences proliferation of human fibroblasts in vitro. It was found that after the treatment with this protein in low doses the cell growth rate increases essentially.
A comparative study of thin carbohydrate specificity of the lectin from the bark of laburnum Laburnum anagyroides (LABA) and fucolectin from asparagus pea Tetragonolobus purpureus (TPA) was performed using inhibition of agglutination of the complex formed by H-active neoglycoprotein and nanoparticles of colloidal gold. Both lectins bound most strongly the H type 2 oligosaccharides comprising O-glycanes; however, TPA was almost unable to discriminate between them. LABA bound more weakly the H type 6 trisaccharide (Fuc alpha 1-2Gal beta 1-4Glc) and difucosyllactose (Fuc alpha 1-2Gal beta 1-4[Fuc alpha 1-3]Glc), a glucoanalogue of the Le(y) antigen, and, even more weakly, the Le(a) pentasaccharide lacto-N-fucopentaose II (Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc). However, LABA did not bind the antigens Le(b), Le(c), and Le(d), very poorly interacted with the terminal Le(x), and somewhat more strongly bound the internal Le(x). The lectin also had a hydrophobic binding site. Both lectins exhibited a cluster effect with polymeric ligands (neoglycoproteins).
A protein with a molecular weight of 70 kDa was isolated from bovine blood serum and purified to a homogenous state. This protein inhibited reversibly the adhesive serum glycoprotein with a molecular weight of 12 kDa, which displayed biological activity at ultralow doses. Amino acid analysis showed that the protein inactivator belongs to the group of prealbumins from vertebrate blood serum. The secondary structure of its molecule was characterized by a considerable number of alpha-helices. The conditions for inactivation of serum glycoprotein were studied. The interaction between the serum glycoprotein and the protein inactivator occurred over a long period of time (1 day). It should be emphasized that the presence of calcium ions was a necessary condition for the inactivation of the serum glycoprotein. The data suggest that inactivation of serum glycoprotein results from the formation of a molecular complex consisting of the protein inactivator and the glycoprotein, which is related to the carbon-protein interaction.
Interaction of fucolectin of perch Perca fluviatilis (PFL) with a set of Lewis antigens was studied by monitoring changes in its tryptophan fluorescence. PFL bound Le(c) (H type 1)-pentasaccharide (Ka = 6.6 x 10(3) M(-1)) and H type 6-trisaccharide (Ka = 2.5 x 10(3) M(-1)); essentially weaker, with Le(b)-hexasaccharide (Ka = = 4.0 x 10(2) M(-1)); and failed to interact with Le(a)-, Le(x)-, and Le(d)-containing oligosaccharides. PFL belongs to a new type of the fucolectins recognizing H-disaccharide Fuc alpha1-2Gal within various antigens, including H type 1/2 and Le(b).
Problems of practical application of fucose-binding lectins as selective histochemical markers and their diagnostical and prognostical value under different pathological states are considered. Special attention is paid to carbohydrate specificity of the most investigated and perspective fucolectins which are already applied in investigation of regularity of cell's functioning and differentiation. It is shown that fucose-specific lectins are useful diagnostic reagents in pathomorphological and clinical investigations.
The regulatory protein was isolated from the eye lens extract by using an early designed scheme including by means of salting-out of proteins by ammonium sulphate, isoelectrofocusing in pH gradient and electrophoresis in PAAG. A high-purity fraction of the regulatory protein was obtained. The localization of the regulatory protein in the rat-eye lens was investigated by means of primary rabbit antibodies obtained within the case study and by FITS-marked secondary antibodies. Cataractogenesis was induced, in vitro, in Wistar rat lenses through adding, to the cultivation medium, hydrogen peroxide (0.5 mM) or calcium chloride (15 mM). The regulatory protein isolated from the bovine eye lens was added alongside with damaging antibodies to the nutrition medium, concentration 10(-12) mg/ml. The lenses were cultivated for as long as 8 days at 37 degrees C. The degree of opacification of lenses was evaluated visually with the help of a lined substrate as well as by spectrophotometry. The studied protein was shown immunohistochemically to be localized in the intercellular space of the lens epithelium in the region of the basic membrane. The cataractogenesis-related research of the regulatory protein was made on rabbit eye lenses, which were cultivated as a whole for as long as 8 days in vitro. Their transparency and morphology were preserved in them in full since they were cultivated in a serum-free nutrition without admixture of any destructive agents. Opacification of lenses was induced in vitro by changing the concentration of calcium ions in the cultivation medium or through adding hydrogen peroxide to the medium. The valuations of the lens opacity degree as observed in different research series and made by visual observation well correlate with the results of spectrophotometry of lenses made after their cultivation. It can be stated that the studied regulatory protein, when added to the cultivation medium, enhances about two-fold the lens transparency versus the lenses cultivated in the catactogenesis-containing medium. Finally, very small doses of the regulatory protein isolated from the bovine eye lens were found to prevent cataractogenesis in rats in vitro. Since the studied regulatory protein was localized by us in the region of epithelium, it can be suggested that its protective action is conditioned by its ability to contribute to regulating the main biological processes occurring in the lens capsule.
A regulatory protein displaying biological activity at ultralow doses was identified in cow's milk. The isoelectric point for this protein falls into the pH range of 4.48-5.59, and the molecular weight does not exceed 10 kDa. A study of the secondary structure detected the predominant presence of beta-structures, especially antiparallel, in the molecule of this regulatory protein as well as the regions described in terms of a statistical globule. It was demonstrated that this protein is located extracellularly in the epithelium of mammary ducts, and that this regulatory protein is present in an active form in whole milk; however, it was detectable neither in dry milk nor in infant formula. The results obtained suggest that the milk regulatory protein characterized in this work was identical to low-molecular-weight serum glycoprotein, one of the proteins studied earlier, displaying biological activity at ultralow doses.