Autoantibodies to 1-adrenergic receptor may play a role in the development of certain cardiovascular system diseases.Synthetic peptide-based enzyme-linked immunosorbent assay (eliSA), traditionally used for the detection of such antibodies, does not allow to obtain adequate data, probably due to the poor similarity of peptide conformations to the spatial structure of the extracellular regions.Aim of the study: to improve the peptide-based eliSA method for the detection of autoantibodies to 1 adrenergic receptor by a separate determination of the igG1, igG2, igG3, igG4, igM and igA autoantibody isotypes.Material and methods: a modification of the eliSA method with peptides corresponding to the sequences of the 1-adrenergic receptor first and second extracellular loops, and secondary antibodies specific to the abovementioned human immunoglobulin isotypes.the method has been tested on a panel of samples obtained from patients with dilated cardiomyopathy (dcM), arrhythmia and coronary heart disease.ResultS: small excess of the signal mean values of individual antibody isotypes have been recorded for some groups of patients in comparison with the group of healthy volunteers.however, these differences were not statistically significant.the proportion of signals «above the 1 ооо «МонА», ул.кастанаевская, 38, стр. 1, Москва, 121108, российская федерация; 2 фГБу «национальный медицинский исследовательский центр кардиологии» Минздрава россии, ул.3-я черепковская, 15а, Москва, 121552, российская федерация Резюме Аутоантитела к 1-адренорецептору могут играть важную роль в развитии некоторых заболеваний сердечно-сосудистой системы.иммуноферментный анализ (ифА) с использованием синтетических пептидов, традиционно применяемый с целью детекции таких антител, не позволяет получать адекватные данные, вероятно, вследствие слабого сходства конформаций пептидов с пространственной структурой внеклеточных участков 1-адренорецептора.Цель исследования.усовершенствовать метод ифА для детекции аутоантител к 1-адренорецептору за счет раздельного определения аутоантител изотипов igG1, igG2, igG3, igG4, igM и igA.Материал и методы.предложена модификация метода ифА с использованием пептидов, соответствующих последовательностям первой и второй внеклеточных петель 1-адренорецептора, и вторых антител, специфических к вышеуказанным изотипам иммуноглобулинов человека.Метод опробован на панели образцов сыворотки крови, полученных от пациентов с дилатационной кардиомиопатией (дкМп), желудочковой аритмией и ишемической болезнью сердца.Результаты. для некоторых групп больных зафиксированы небольшие превышения средних значений сигналов отдельных изотипов антител по сравнению с группой здоровых добровольцев, однако эти отличия не являются статистически значимыми.доля сигналов «выше уровня нормы» в группе больных дкМп составила 21% для изотипа igG1 и от 6 до 16% для остальных изотипов, что весьма далеко от частоты встречаемости аутоантител к 1-адренорецептору (70-80%), наблюдаемой у больных дкМп при использовании функциональных методов анализа.в других группах доля таких сигналов не превысила 30% ни для одного изотипа антител.

The role of beta2-integrins CD11b/CD18 and CD 11c/CD 18 in adhesion and migration of leukocytes on fibrinogen was studied. The monoclonal antibodies against CD11b inhibited the spontaneous adhesion of monocytic THP-1 cells on fibrinogen, whereas antibodies to CD11c more effectively inhibited the adhesion stimulated by chemokine MCP-1. By the RNA-interference method the clones of THP-1 with reduced expression of CD11b and general beta2-subunit CD18 were obtained. MCP-I stimulated the adhesion to fibrinogen of THP-1 cells of wild-type and mutant cells with reduced expression of CD11b (THP-1-CD11b-low), but not of cells with low expression of CD18 (THP-1-CD18-low). THP-1-CD18-low cells were also characterized by the impaired chemotaxis in presence of MCP-1. The data obtained suggest that spontaneous cell adhesion to fibrinogen is mediated to a greater extent by CD11b/CD18 integrins, while chemokine-stimulated adhesion and migration is mostly dependent on CD11c/CD18 molecules.

In the present study, human keratinocytes and dermal papilla cells were labeled to investigate their behaviour after intradermal transplantation. Cells were transduced by lentiviral vectors that bore marker gene encoding green fluorescent protein (copGFP) or red fluorescent protein (DsRed). A portion of transgene expressing cells was evaluated by flow cytometry. Genetic constructions that we used provided high level (> 95 %) of transduction of hair follicle cells. In vitro transduced cells were injected under the epidermis of human skin fragments, and these fragments were then transplanted under the skin of immunodeficient mice. Injected epidermal keratinocytes were found, mainly, in hair follicles and partially in a zone of interfollicular epidermis, while dermal papilla cells were found in papilla derma. The results of the present research show that the chosen genetic constructions obtained on a basis of human immunodeficiency lentivirs are capable of effective and stable transduction of human skin cells. Injected cells survived and were found in the corresponding structures of the skin.

The effect of the suppression of expression of the actin-binding protein caldesmon on the motility of nonmuscle cells has been studied. A more than fivefold decrease in the content of this protein in cells by RNA interference led to the disturbance of the formation of actin stress fibrils and acceleration of cell migration to the zone of injury of the monolayer. A stimulation of stationary cells by serum induced a more than 1.5-fold accumulation of stress fibrils only in control cells but not in caldesmon-deficient cells. Similarly, the accumulation of actin filaments was observed in actively migrating cells of only wild type but not in cells with a low caldesmon content. These changes occurred mainly at the leading edge of the migrating cell where the distinct structure of actin filaments was not seen in the absence of caldesmon. It was assumed that caldesmon inhibits cell migration due to the stabilization of actin in filaments and a decrease in the dynamics of monomeric actin at the leading edge of the migrating cell.

Effects of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (I), 3alpha-hydroxy-5alpha-cholest-8(14)-en-15 one (II), 3beta-hexadecanoyloxy-5alpha-cholest-8(14)-en-15-one (III), 3alpha-hehadeeanoyloxy-5alpha-cholest-8(14)-en-15-one (IV), 3beta-acetoxy-5alpha-cholest-8(14)-en-15-one (V), 3alpha-acetoxy-5alpha-cholest-8(14)-en-15-one (VI) on cholesterol metabolism in hepatoma Hep G2 cells were studied. Compound III slowly bind to Hep G2 cells followed by internalization and metabolic transformation (at a concentration of 30 microM the total binding of compound III was (3.9 +/- 0.4) nmol per 1 mg of cell protein for 24 h incubation). Compound I depressed and compound III stimulated the uptake of low density lipoproteins radiolabeled with oleyl cholesteryl ether [14C-CE]LDL (58% and 149% from control). Compounds I and II inhibited cholesterol biosynthesis from [14C]acetate (with IC50 values of 4.0 +/- 0.7 and 8.0 +/- 1.5 microM). Effects of compounds V and VI were less potent; compounds III and IV were inactive. Compound II activated cholesterol acylation, estimated by incorporation of [14C]-oleic acid into cholesteryl esters (170% from control at a concentration of 30 microM). The results indicate correlation between polarity of the compound and its ability to regulate cholesterol metabolism in Hep G2 cells.

Expression of urokinase in murine and rat cells was performed by two recombinant constructs, one containing cDNA and the other--hybrid (cDNA/genome) variant of human urokinase gene conserving 7 introns of 10, in the eukaryotic retrovirus vector pPS-3-neo. DNA of both constructs was introduced into packaging cell line psi 2 by a standard Ca-phosphate transfection technique. Infection of mouse and rat fibroblasts BALB/c 3T3 and Rat I with virus particles, produced by transfected psi 2 cells, led to an integration into the host genome of one or two recombinant proviral copies. Stable expression and secretion into the culture medium of glycosylated high molecular weight human urokinase was observed for both cell types. For the hybrid gene construct, precise excision of intervening sequences was shown during transferring of genetic material from packaging to recipient cells.

Poly(A)+-RNA from human kidney and human embryonal lung fibroblasts fractionated by sucrose gradient centrifugation was translated in Xenopus oocytes. Assay for plasminogen-dependent fibrinolytic activity detected synthesis of secreted plasminogen activator and revealed the active fraction of poly(A)+-RNA with a sedimentation coefficient of approximately 23S. Translation products of the active fraction were immunoadsorbed by antiurokinase monoclonal antibodies immobilized on sepharose. Gel electrophoretic analysis of the protein products showed that the 23S fraction of poly(A)+-RNA from human kidney contains mRNA for single-chain urokinase-type plasminogen activator with apparent molecular weight of approximately 50 kDa.