Myosin light chain kinase colocalizes with nonmuscle myosin IIB in myofibril precursors and sarcomeric Z-lines of cardiomyocytes.

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility involving actin and myosin II. MLCK is widely present in vertebrate tissues including the myocardium. However, the role of MLCK in cardiomyocyte function is not known. Previous attempts to gain insight into possible roles and identify potential molecular partners were disappointing and equivocal due to cross reactivity of early antibodies with striated muscle MLCK, which has a different genetic locus and a divergent amino acid sequence from the above mentioned enzyme. Using an immunofluorescence approach and a panel of antibodies directed against MLCK, cytoskeletal, and sarcomeric proteins, we localized MLCK to myofibril precursors and Z-lines of sarcomeres in embryonic and adult cardiomyocytes. The same structures contained nonmuscle myosin IIB implicating this protein as a possible target of MLCK. Our results suggest a role for MLCK in cardiomyocyte differentiation and contraction through regulation of nonmuscle myosin IIB.

[Phenotypes of smooth muscle cells in carotid arteries in Takayasu's disease]. / Fenotipy glsadkomyshechnykh kletok v sonnykh arteriiakh pri bolezni Takaiasu.

The phenotype of smooth-muscle cells (SMC) of carotid arteries in health and Takayasu's disease were studied using immunofluorescence. In contrast to unaffected carotid artery, medial SMC were represented by heterogenic cell population. Many SMC in this disease expressed fibronectin with ED-A sequence, contained cytokeratin 8 and were deprived of h-caldesmon. Similar phenotypic characteristics are typical for SMC of embryonal phenotype. SMC with this phenotype are observed in the thickened intima of the carotid arteries in Takayasu's disease. It is supposed that SMC precursors in arterial media are activated in this disease, migrate into subendothelial layer and are responsible for the thickening of the intima.

[Isoproterenol induced changes of protein expression and myocardial ultrastructure]. / Izmeneniia ékspressii belkov i ul'trastruktury serdechnoi myshtsy pod vliianiem izoproterenola.

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.

[Effect of adriamycin on expression and content of myocardial structural and regulatory proteins]. / Izmeneniia ékspressii strukturnykh i reguliatornykh belkov v miokarde pri deistvii adriamitsina.

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.

[Functional properties and intracellular localization of high molecular weight isoforms of ligh chain myosin kinase]. / Funktsional'nye svoistva i vnutrikletochnaia lokalizatiia vysokomolekuliarnoi izoformy kinazy legkikh tsepei miozina.

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.

[Clinical course of restenosis after carotid endarterectomy]. / Klinicheskoe techenie restenozov posle karotidnoi éndarteriéktomii.

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.

[Structural changes in the collagen fibers of the connective tissue in chronic inflammation in the periodontium]. / Izmeneniia stroeniia kollagenovykh volokon soedinitel'noi tkani pri khronicheskom vospalenii v periodonte.

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.

[Phenotypic characterization of cells and structure of the extracellular matrix in pleomorphic adenoma of the salivary glands]. / Fenotipicheskie kharakteristiki kletok i stroenie ékstratselliuliarnogo matriksa v pleomorfnoi adenome sliunnykh zhelez.

Phenotypic characteristics of cells and extracellular matrix (ECM) of 14 salivary gland pleomorphic adenomas were studied by indirect immunofluorescence. All cells in the epithelial structures expressed cytokeratin 8, the majority of cells produced vimentin, laminin and IV type collagen. Cells of myxoid areas expressed vimentin: type IV collagen was observed in the form of fibrillar structures. Fibrous areas were characterized by vimentin and cytokeratin 8 expression; types I, III, IV collagens and fibronectin with ED-A sequence were present in ECM. Terms "epithelial structures", "epithelial" and "myoepithelial" cells in relation to pleomorphic adenoma may be used only relatively as it is mainly represented by cells with features of both epithelial and mesenchymal phenotype. Characteristics of ECM synthesized in various structures of pleomorphic adenoma may serve an additional criterion of cell transformation.

[Electrophoretic characteristics of integrin++ beta1 subunit from human smooth muscles]. / Electroforeticheskoe povedenie beta1 sub''edinitsy integrina iuz gladkikh myshts cheloveka.

Total fraction of beta 1 integrin family was isolated from human smooth muscle by affinity chromatography on immobilized anti-beta 1 monoclonal antibodies. SDS-PAGE analysis and subsequent immunoblotting demonstrated that integrin samples contain unknown before high molecular mass (205 kD-nonreduced and 230 kD-reduced) material immunologically related to beta 1 integrin subunit. 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. Reduction of electrophoretic samples with dithiothreitol led to the removal of the major part of the beta 1 immunoreactive material from 205 kD to 130 kD region, indicating a disulfide nature of B1 integrin subunit dimer. The 230 kD protein turned out to be an only partly reduced beta 1 integrin disulfide bonded dimer. Possible in vivo existence of the disulfide bonded dimer and oligomer integrin forms is discussed.

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.

[Structure of connective tissue in inflammatory-sclerotic processes in the periodontium]. / Osobennosti stroeniia soedinitel'noi tkani pri vospalitel'no-scleroticheskikh protsessakh v periodonte.

Two main types of extracellular matrix distribution was distinguished on the basis of immunohistochemistry of the periodontium connective tissue in various inflammatory-sclerotic processes. The 1st type was characterized by a significant reduction of interstitial collagen, moderate content of type V collagen and high content of fibronectin (in case of the exacerbation). The 2nd type was characterized by a high content of type V collagen, increased content of interstitial collagen and moderate amount of fibronectin (in case of reparative sclerosis). An inversion of the relation between the I and III collagen types in favour of the latter, high level of type V collagen and permanent presence of fibronectin were characteristic for the scar tissue. These properties of scar connective tissue indicate its immaturity and permanent activity.

[Sclerotic changes in the glomeruli in glomerulonephritis in children (immunohistochemical study]. / Skleroticheskie izmeneniia v klubochkakh pri glomerulonefritakh u detei (immunogistokhimicheskoe issledovanie).

The structure of the extracellular matrix (ECM) in the kidney glomeruli in different variants of glomerulonephritis (GN) was studied using immunofluorescence. Mesangial sclerosis and synthesis of the interstitial collagen, type I and III, were found to be a peculiar feature of mesangioproliferative and mesangiocapillary GN. Location of sclerosis predominantly in the basal membranes of the peripheral capillary loops was typical for membranous GN. Restructuring of ECM in minimal changes and focal-segmentary glomerulosclerosis is limited by an increase of fibronectin in the mesangium and peripheral capillary loops of the glomeruli. The development of pronounced sclerosis and hyalinosis of glomeruli as well as the formation of the crescents are the most marked ECM restructuring signs regardless of GN form.

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.

[The immunohistochemical study of suppurative wounds in rats following the application of collagenase from the crab Paralithodes camtschatica]. / Immunogistokhimicheskoe izuchenie gnoinykh ran u krys posle applikatsii kollagenazy kraba 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.

[The phenotypes of arterial smooth-muscle cells during ontogeny and in atherosclerotic plaques]. / Fenotipy gladkomyshechnykh kletok arterii v protsesse ontogeneza i v ateroskleroticheskikh bliashkakh.

Smooth-muscle cell (SMC) myosin expression, SMC alpha-actin, h-caldesmon and calponin expression were studied in developing SMC of embryonic aorta as well as in adult human aorta and coronary arteries. It was found that ontogenesis is associated with SMC phenotypic modulations. In 8-23-week embryos SMC express SMC myosin and alpha-actin. In adult humans arterial SMC express all the markers studied. Normal subendothelium contains a heterogeneous SMC population. SMC heterogeneity is most marked in atherosclerotic plaques in the form of clusters of homogeneous cells different by expression of contractile system proteins. It is suggested that SMC heterogeneous population in atherosclerotic plaques may arise due to proliferation of phenotypically different precursors cells.

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.

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.

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.

[Preparation and use of monoclonal antibodies against seal alkaline phosphatase]. / Poluchenie i primenenie monoklonal'nykh antitel proteiv shchelochnoi fosfatazy tiulenia.

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.