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B V Shekhonin

Publications and source records attributed to B V Shekhonin.

At least 19 recordsLinked to original sources
PubMed2002

[Isoproterenol induced changes of protein expression and myocardial ultrastructure].

AIM: To elucidate alterations in myocardial ultrastructure and protein expression caused by isoproterenol. METHODS: Biochemical, immunohistochemical and electron microscopic studies of rat myocardium were carried out 2 hours and 3 weeks after single injections of isoproterenol (50 and 10 mg/kg). Relative content of myospecific proteins (KRP - kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, and myosin light chain kinase - MLCK) and extracellular matrix protein, fibronectin, was determined by immunoblotting. RESULTS: In 2 hours after injection of 50 mg/kg of isoproterenol destruction of some cardiomyocytes, contracture of myofibrils, and mild edema of intercellular space occurred; the content of KRP decreased by l6%, and that of tubulin, vinculin and fibronectin - by 27-29%. Reduced level of these proteins and also of MLCK persisted until 3 weeks after injection and was associated with altered cardiomyocyte ultrastructure. Glycogen granules were sparse, mitochondria contained arrow-like inclusions characteristic for calcium overload, huge mitochondria connected by specialized intermitochondrial contacts were present. Enlarged intercellular space contained areas of fibrosis with increased amount of type I and II collagens and fibronectin. Lower dose of isoproterenol (10 mg/kg) did not cause noticeable damaging action in the acute period, but in 3 weeks thickening of extracellular matrix occurred accompanied by increases of KRP and tubulin contents (by 26-32% compared with control level). Similar rise in expression of these proteins, and also of MLCK was observed after addition of isoproterenol to culture of chicken cardiomyocytes. CONCLUSION: These results indicate that even single injection of isoproterenol causes long lasting structural alterations in cardiac muscle accompanied by increased expression of extracellular matrix proteins as well as sarcoplasmic proteins apparently involved in the hypertrophic response of the cardiomyocytes.

Adrenergic beta-Agonists↗
PubMed2002

[Effect of adriamycin on expression and content of myocardial structural and regulatory proteins].

The biochemical and morphological study of the cytoskeleton and extracellular matrix of rat heart was carried out after single injection of adriamycin (2.2 or 0.44 mg/kg). Hearts were taken for the study after 2 hours and 3 weeks after injection. The light and electronic microscopy, immunohistochemical determination of type I, III and IV collagens and fibronectin using specific antibodies were implied for morphological study; electrophoresis and immunoblotting were implied for the determination of the content of some proteins of cardiomyocytes (KRP or telokin, desmin, tubulin and vinculin), and extracellular matrix (fibronectin) and vascular smooth muscle cells (MLCK, myosin light chain kinase). Adriamycin injection in the dose 2.2 mg/kg which is close to therapeutic and known to alter intracellular membranes approximately in the half of cardiomyocytes, did not influence the relative volume and structure of collagen network but distinctly reduced the density of fibronectin-distribution. The content of tubulin, fibronectin, MLCK and KRP was significantly decreased by 18-24%, while contents of desmin and vinculin were changed insignificantly. After 3 weeks, an increased density and extension of collagen network indicating the development of diffuse fibrosis were observed. Contents of tubulin and KRP were increased above control level by 50 and 20%, respectively. Similar hyperrestitution of tubulin, fibronectin and KRP content by 15-25% was determined after smaller dose of adriamycin (0.44 mg/kg). Only content of MLCK out of proteins studied remained at lower level in both groups by 25-34%. Isolated chick embryo cardiomyocytes subjected to adriamycin responded by increased level of KRP expression by 20% in 4 days while the level of tubulin expression remained unchanged. Results showed that damage of cardiomyocytes and extracellular matrix after single injection of adriamycin in the dose close to therapeutic was followed by increased expression of some proteins of cytoskeleton and extracellular matrix. KRP seems to play active role in this reparative response while the steadily reduced level of MLCK expression may disturb the control of coronary vessels.

Animals↗
PubMed2000

[Functional properties and intracellular localization of high molecular weight isoforms of ligh chain myosin kinase].

The vertebrate genetic locus, coding for a Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK), the key regulator of smooth muscle contraction and cell motility, reveals a complex organization. Two MLCK isoforms are encoded by the MLCK genetic locus. Recently identified M(r) 210 kDa MLCK contains a sequence of smooth muscle/non-muscle M(r) 108 kDa MLCK and has an additional N-terminal sequence (Watterson et al., 1995. FEBS Lett. 373 : 217). A gene for an independently expressed non-kinase product KRP (telokin) is located within the MLCK gene (Collinge et al., 1992. Mol. Cell. Biol. 12 : 2359). KRP binds to and regulates the structure of myosin filaments (Shirinsky et al., 1993. J. Biol. Chem. 268 : 16578). Here we compared biochemical properties of MLCK-210 and MLCK-108 and studied intracellular localization of MLCK-210. MLCK-210 was isolated from extract of chicken aorta by immunoprecipitation using specific antibody and biochemically analysed in vitro. MLCK-210 phosphorylated myosin regulatory light chain and heavy meromyosin. The Ca(2+)-dependence and specific activity of MLCK-210 were similar to that of MLCK-108 from turkey gizzard. Using sedimentation assay we demonstrated that MLCK-210 as well as MLCK-108 binds to both actin and myosin filaments. MLCK-210 was localized in smooth muscle cell layers of aortic wall and was found to co-localize with microfilaments in cultured aortic smooth muscle cells.

Animals↗
PubMed1998

[Clinical course of restenosis after carotid endarterectomy].

Angiologic and neurologic examinations, ultrasonic dopplerography were performed in 89 patients after operation of carotid endarterectomy. Period of observation was 6-182 months after operation (61 months on the average). Positive clinical effect (absence of disorders of cerebral circulation-DCC) was achieved in 91% of cases. The frequency of both repeated transitory ischemic attacks and the strokes was 9%, the best frequency being in patients with initially asymptomatic course. It was found that the degree of restenosis directly correlated with frequency of neurologic symptom development: there were no repeated DCC in normal state of the operated artery. Meanwhile DCC frequency was less than 4% in cases with the degree of restenosis less than 60% and was equal to 15.8% in more than 60% restenosis. DCC were more frequently found in occlusion of internal carotid artery. Rather favourable course of restenosis of carotid artery was also conditioned by low content of the plaques dangerous in terms of embolism. Progression of atherosclerotic damages in other parts of extracranial arteries promoted clinical manifestation of cerebral ischemia. Moreover, negative neurologic dynamics developed in 7% of the patients with restenosis less than 60%, and in 13.8% of cases with restenosis more than 60%. The conclusion was made about efficiency of carotid endarterectomy in patients with atherosclerotic damages of carotid arteries. For prevention of repeated DCC it is recommended both to perform conservative therapy after the operation and to examine dynamically the patients using ultrasonic dopplerography beginning 6 months after the operation.

Adult↗
PubMed1998

[Structural changes in the collagen fibers of the connective tissue in chronic inflammation in the periodontium].

Data of examinations of the periodontium during chronic inflammations are analyzed. Immunohistochemical studies revealed that the connective-tissue cicatrix forming in the course of reparative regeneration of the periodontium is immature and incapable of performing mechanical functions. The authors suggest that the above regeneration abnormalities lead to a relapsing occlusion injury of the periodontium and progress of the inflammatory sclerotic process. Perspective trends of the patho- and morphogenetic research of chronic periodontitis are outlined, as are the probable theoretical approaches to development of methods for correction of such abnormalities.

Adult↗
PubMed1996

Beta 1 integrin subunit dimerization via disulfide bonds.

Integrins of the beta 1 family were isolated from human smooth muscle. SDS-PAGE analysis and subsequent immunoblotting demonstrated that integrin samples contain a protein immunologically related to beta 1 integrin subunit with the previously undescribed apparent molecular mass 205 kD. One-dimensional peptide mapping showed that the 205 kD protein is not a novel beta 1 related integrin subunit, but a beta 1 integrin subunit dimer. After reduction the major part of the beta 1 immunoreactive material migrated from the 205 kD to 130 kD region, indicating that beta 1 integrin subunit dimers were formed via disulfide bonds. When electrophoretically pure beta 1 monomer and dimer forms were analized it was found that during SDS-PAGE about 30% of beta 1 integrin subunit monomers were organized into dimers while approximately 70% of the beta 1 dimer form was partly disrupted into monomers. It was suggested that this steady-state process is a result of a reversible reaction between intra- and intermolecular disulfide bonds. Possible in vivo dimerization of integrins via disulfide bonds is discussed.

Chromatography, Affinity↗
PubMed1994

Immunohistochemical study of purulent wounds treated with King crab collagenase.

Immunohistochemical study of tissues from purulent wounds in rats after treatment with the collagenase isolated from the King crab Paralithodes camtschatica was undertaken. The enzymotherapy resulted in a rapid and efficient removal of necrotic debris. It was accompanied by fibrin elimination from the wound bed and subsequent formation of new capillaries. Cellular fibronectin with ED-A sequence was identified in the newly formed granulation tissue, which points to its active synthesis in situ. Polyclonal antibodies against two isozymes of the crab collagenolytic protease were obtained. By their use it was shown that, after application of the collagenase, both isozymes accumulated in fibrin deposits at the wound bed but did not penetrate adherent granulation tissue.

Animals↗
PubMed1993

[The immunohistochemical study of suppurative wounds in rats following the application of collagenase from the crab Paralithodes camtschatica].

Immunohistochemical study of tissues of purulent wounds in rats after application of the collagenase isolated from the king crab Paralithodes camtschatica has been undertaken. The enzyme therapy resulted in a rapid and efficient removal of necrotic debris. It was accompanied by fibrin elimination from the wound bottom and subsequent formation of new capillaries. Cellular fibronectin with ED-A sequence was identified in the newly formed granulation tissue, which points to its active synthesis in situ. Detection of type I collagen in granulation tissue revealed that wound treatment with crab collagenase had no impact on the development process of the tissue. Polyclonal antibodies against two isozymes of crab collagenolytic protease were obtained. It was shown that after application of both isozymes of the collagenase were accumulated in fibrin deposits at the wound bottom but not penetrated in adherent granulation tissue. These processes underlie the therapeutic effect of the crab collagenase.

Animals↗
PubMed1992

Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin.

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of alpha-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8-10 and 20-22 weeks of gestation express alpha-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of alpha-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.

Adolescent↗
PubMed1992

Monoclonal antibody to alkaline phosphatase from the intestinal mucosa of the harp seal, Phoca groenlandica.

1. Hybridoma secreting a monoclonal antibody APP.1 to the harp seal alkaline phosphatase (A1Ph) was obtained by fusing murine myeloma Sp 2/0 cells with the splenocytes of BALB/c mice immunized with purified isozyme K. 2. The antibody has no effect on the enzyme activity and shows a high affinity for harp seal A1Ph (KD = 8.5 x 10(-10) M). The antibody has similar affinities for the AlPh of harp seal, fur seal, common seal and deer. 3. The antibody APP.1 was coupled to Sepharose and employed in chromatographic purification of the harp seal intestinal AlPh. Alkaline phosphatase isolated on this immunosorbent has a spec. act. of 20,800 units per mg of protein. 4. The antibody-enzyme complex gives an excellent immunocytochemical labeling of tissue sections, cell cultures and smears.

Alkaline Phosphatase↗
PubMed1991

Expression of cytokeratin 8 in human aortic smooth muscle cells.

An immunofluorescence method was used to study the expression of cytokeratin 8 in human aortic smooth muscle cells (SMCs) during prenatal development and in atherosclerotic plaques. Aortic SMCs from a 10-wk-old fetus contained cytokeratin 8 in additional to vimentin and a small amount of desmin, whereas, in the cells from a 25-wk-old fetus, cytokeratin 8 was not detected. Cytokeratin 8 was found in the SMCs from intimal thickenings, fatty streaks, and atherosclerotic fibrous plaques. Clusters of cytokeratin 8-positive cells were more abundant in rather advanced lesions (fibrous plaques) that contained at least some amount of lipid. Expression of cytokeratin 8 in the cells of human atherosclerotic lesions probably reflects general rearrangement of gene expression in the intimal cells.

Adult↗
PubMed1991

[Preparation and use of monoclonal antibodies against seal alkaline phosphatase].

Monoclonal antibodies (termed as APP.1 and related to subclass IgG1) against seal alkaline phosphatase, have been obtained. APP.1 did not influence the enzymatic activity of alkaline phosphatase. The dissociation constant for the APP.1 interaction with Greenland seal alkaline phosphatase was equal to 8.5 x 10(-10) M. It was found that APP.1 interact with intestinal isoenzymes of common and fur seal, calf and deer alkaline phosphatases. An APP.1 complex with seal alkaline phosphatase was obtained and successfully applied in immunoenzymatic analysis. The use of this complex made it possible to diminish the limit of detectability of antibodies against peptide fragments of HIV-1 and HIV-2 proteins. Moreover, this complex allowed the identification of cytokeratin-8 and vimentin in human kidney slices and embryonic fibroblast-like cells, respectively.

Alkaline Phosphatase↗
PubMed1990

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development.

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.

Aorta↗
PubMed1990

Visualization of apo B, fibrinogen/fibrin, and fibronectin in the intima of normal human aorta and large arteries and during atherosclerosis.

Apolipoprotein B (apo B), fibrinogen/fibrin, blood platelets, factor VIII-related antigen of the blood coagulation system, and smooth muscle cells (SMC) were identified in the intima of normal and atherosclerotic human aorta and large arteries by the indirect immunofluorescence technique. Fibrinogen/fibrin was revealed by a monoclonal antibody (monAb) against the C-terminal region of human fibrinogen A alpha-chain. Fibronectin was visualized by monAb to the cellular form and against an epitope shared by different fibronectin subunit variants. In normal intima, fatty streaks, small amounts of fibrinogen/fibrin together with large amounts of apo B were observed. Fibronectin detected by two types of monAb was not found in extracellular matrix (ECM), whereas cellular fibronectin encircled SMC. According to the data obtained, fibrinogen/fibrin accumulates in plaques as a result of intramural thrombus incorporation, blood insudation, intramural haemorrhage, and in or around cells, apparently macrophages.

Aged↗
PubMed1990

Immunofluorescent identification of fibronectin and fibrinogen/fibrin in experimental myocardial infarction.

Distribution of fibrinogen/fibrin and fibronectin in regions of experimental myocardial infarction were studied by the immunofluorescence technique. Distinct from normal myocardium 3 and 12 to 24 h after coronary artery ligation infiltration of necrotized cardiomyocytes by fibrinogen/fibrin and plasma fibronectin was detected. Fibrinogen/fibrin and plasma fibronectin constitute "primary matrix" of granulation tissue. On the third day after experimental infarction, synthesis of cellular fibronectin begins. Its content in the extracellular matrix (ECM of granulation tissue significantly increases on days 7 to 15. The amount of fibronectin in the ECM of developing scar tissue dramatically decreases 30 days after infarction, Fibrinogen/fibrin was continually identified in granulation tissue in zones of myocardial infarction. However, its amount in the ECM of developing scar tissue gradually decreased.

Animals↗
PubMed1990

[Apo B, fibrinogen-fibrin and fibronectin in the intima of the normal human aorta and large arteries and in atherosclerosis].

Apo B, fibrinogen/fibrin, fibronectin, thrombocytes, factor VIII of the blood coagulation and smooth muscle cells (SMC) were identified by the immunofluorescence method in the intima of aorta and big arteries under normal conditions and in atherosclerosis. Monoclonal antibodies (MCA) against C-end fragments of A alpha-fibrinogen chain were used in the study of fibrinogen/fibrin. MCA reacting with plasma fibronectin and those reacting with A-area of the polypeptide chain specific for the cell fibronectin were used for the identification of fibronectin. Small amount of fibrinogen/fibrin, no fibronectin in the extracellular matrix and the cell fibronectin around SMC were observed on the normal intima and lipid strip in spite of the presence of Apo B. The results indicate that fibrinogen/fibrin is accumulated in the plaques due to the incorporation of the wall thrombi, insudation from the blood plasma, intramural haemorrhages as well as around cells, presumably macrophages.

Adult↗
PubMed1989

Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent.

Different fibronectin (FN) variants arise from the single gene transcript alternatively spliced in a tissue-specific manner (Hynes, R. O. 1985. Annu. Rev. Cell Biol. 1:67-90; Owens, R. J., A. R. Kornblihtt, and F. E. Baralle. 1986. Oxf. Surv. Eurcaryotic Genes. 3:141-160). We used mAb IST-9, specific for extra domain A (ED-A) FN sequence, and cDNA probe to ED-A exon to determine whether ED-A is present in FN synthesized by vascular smooth muscle cells (SMCs) and, if so, whether expression of ED-A is SMC phenotype dependent. ED-A-containing FN (A-FN) was not revealed in tunica media of human arteries and normal rat aorta by immunofluorescence and immunoblotting techniques. A cDNA probe to ED-A exon did not hybridize with RNA isolated from human aortic media. A positive reaction with IST-9 was observed in (a) diffuse intimal thickening and atherosclerotic plaque from human arteries; (b) experimentally induced intimal thickening in rat aorta; and (c) cultured vascular SMCs. A-FN mRNA was present in the RNA preparation from human aortic intima as judged by hybridization with cDNA probe to ED-A. On the other hand, an mAb interacting with an epitope common for all FN variants revealed FN in both intima and media of human arteries and in the normal rat aorta. A cDNA probe to a sequence shared by all FN variants hybridized with RNA from both intima and media of human aorta, though the level of expression was higher in intima. The data suggest that ED-A exon is omitted during splicing of the FN mRNA precursor in medial SMCs while the expression of A-FN is characteristic of "modulated" SMCs--those of intimal thickenings, of atherosclerotic lesions, and growing in culture.

Animals↗