Expression of adaptor protein Ruk/CIN85 isoforms in cell lines of various tissue origins and human melanoma

Aim: Development of monoclonal and polyclonal antibodies against recombinant GST-fused proteins including correspondingly N- and C-terminal parts of Ruk/CIN85 adaptor protein. Analysis of Ruk/CIN85 expression patterns in cell lines of various tissue origins and human melanoma. Methods: Recombinant GST-fused fragments of Ruk/CIN85 were expressed in bacterial system and affinity purified. Monoclonal antibodies against SH3A domain of Ruk/CIN85 were produced using hybridoma technique. The specificity of generated antibodies was examined by ELISA. Polyclonal antibodies against C-terminal coiled-coil region of Ruk/CIN85 were affinity purified from serum of immunized rabbit. Expression patterns of Ruk/CIN85 isoforms and their subcellular localization in cell lines of various tissue origins and human melanoma samples were analyzed by immunoblotting, immunoprecipitation and immunofluorescence microcopy. Results: Ruk/CIN85 is ubiquitously expressed SH3-containing adaptor/scaffold protein which plays important roles in signalling processes. N-terminal half of Ruk/CIN85 molecule, including three SH3 domains, and its C-terminal coiled-coil region were used as antigens to produce monoclonal and polyclonal antibodies, respectively. Hybridoma cell lines secreting monoclonal antibodies (mAbs) to SH3 fragment of Ruk/CIN85 were established. One of the mAbs was extensively characterized and designated as MISh-A1. It was shown that this mAb recognizes an epitope, which resides within first SH3A domain. Polyclonal anti-Ruks Abs affinity purified from serum of immunized rabbit specifically recognized main Ruk/CIN85 isoforms, both endogenous and recombinant, in lysates of HEK293 cells. Notably, produced Abs did not cross-react with CD2AP, the member of the same family of adaptor/scaffold proteins. Multiple molecular forms of Ruk/CIN85 with apparent molecular weights of 130, 80–85, 70–75, 50–56, 34–40 and 29 kD were detected in cell lyzates of NIH3T3, Cos1, L1210, HEK293, Ramos, HeLa S3, MDCK, C6, A549 and U937 using anti-Ruk antibodies. Oligomerization between p85 and p50–56 forms of Ruk/CIN85 was revealed in C6 and NIH3T3 cells, but not in HeLa S3 and HEK293 cells by immunoprecipitation using MISh-A1 antibody following anti-Ruk Western-blot analysis. Using immunofluorescent microscopy and anti-Ruk antibodies, endogenous Ruk-variates were found mostly in cytoplasm of C6, NIH3T3, HEK293 cells and at lower level — in nuclei. Conclusion: Patterns of Ruk/CIN85 molecular forms expression are cell-specific and determined by cellular context. Assembly of oligomeric complexes between p85 and p50–56 Ruk/CIN85 isoforms in C6 and NIH3T3 cells but not in HeLa S3 and HEK293 cells may reflect their specific biological roles in different cell lines. High level of full-length Rukl/CIN85 form expression was revealed in extracts of human melanoma samples. Abs described in this paper may prove useful in future studies of Ruk/CIN85 expression and function in normal and transformed cells.Цель: получение моноклональных и поликлональных антител против рекомбинантных GST-коныогированных форм белков, включающих соответственно N-концевую и С-концевую части адаптерного белка Ruk/CIN85. Авалю профилей экспрессии изоформ Ruk/CIN85 и юс субклеточной локализации в клеточных линиях различного тканевого проасхождешя н образцах меланомы человека. Методы: рекомбинантные GST-конъюгнрованные фрагменты Ruk/CIN85 (GST-3SH3/CIN85 и GST- Ruk ) экспрессировали в бактериальной системе с последующей аффинной очисткой на глутатнон-сефарозе. Для получения моноклональных антител против 3SH3 фрагмента С IN85 использовали гнбридомную технологию. Поликлоналыеле антитела против С-конневого суперсинралнзоваииого района Ruk очищали из сыворотки иммунизированного кролика аффинной хроматографией на Ruk -сефарозе. Профази экспрессии изоформ Ruk/CTN85, их олигомеризацию и субклеточную локализацию изучали метолами Вестерн-блотанализа, нммувопрецнпнгацнн и иммунофлуоресиентной микроскопии. Результаты: Ruk/CIN85, S НЗ-содержащий адаптерный “стеллажный” белок, преет важную роль в сшив мытых процессах клеток. N- конневую часть молекулы CIN85, включающую 3 SH3 домена, н С-конценой суперспирализокаиный райе» использовали в качестве антигенов для получения моноклональных и нолик.тональных антител, соответственно. Получены гибрнломы. секретнру юнше моноклональные антитела к 3SH3-фрагменту. Показано, что одно из этих антител (MISh-Al) специфически узнает эпитоп, находящийся в первом $НЗАдомеие адаптерного белка. Поликлональные анти-Ruk антитела, аффинно очищенные из сыворотки иммунизированного кроля, снецифическм узнавати основные изоформы Ruk/CIN85, как эндогенные, так и рекомбинантные, в лизатах клеток НЕК293. Важно, что полученные антитела не реагировази перекрестно с CD2AP, представителем этого же семейства атаптерных белков. Множественные молекулярные формы Ruk/CIN85 с кажущейся молекулярной массой 130. 80-85, 70-75, 50-56, 34-40 and 29 кДа были детектированы в лизатах клеток NIH3T3, Cost, 1.1210, НЕК293, Ramos, HeLa S3, MDCK, Сб, A549 и U937 с Rak антител. Олигомеризацию между р85 и р50-56 формами Ruk/CIN85 выявили в клетках С6 и NIH3T3, во не в HeLa S3 и НЕК293, с помощью антитела MISh-AI и последующего анги-Rak Вестерн-блот анализа. Иммунофлу оресиентной микроскопией с использованием анти-Ruk антител эндогенные варианты Ruk выявляли прешущественно в цитоплазме клеток С6, NIH ЗТЗ, НЕК293 и в более низкой степени в ядре. Высокий уровень экспресс» полноразмерной формы RA/C1N85 выявлен в образцах меланомы человека. Выводы: профили экспрессии множественных эндогенных вариантов Ruk/CIN85 и способность к формированию олигомерных комніексов между различными и зоформами являются характерными для каждой исследованной линии клеток. Не исключено, что выявленные особенности могут определять специфическую биологическую значимость изоформ Ruk/CEN85 в различных типах клеток в зависимости от клеточного контекста. Следствием высокого уровня экспрессии полноразмерной формы Rnk/CIN85 в образцах меланомы человека может быть нарушение согласованного контроля процессов пролиферации и апоптоза в трансформированных ме.таноцитах. Антитела, описанные в этой работе, могут быть использованы для изучения экспрессии и функций Ruk/CIN85 в опухолях человека

Cardiac tumors: analysis of surgical treatment

Aim. To analyze the preoperative status, intraoperative tumor characteristics and further clinical manifestations in patients after surgery for a cardiac tumor.Material and methods. The study included 54 patients who were operated on for a heart tumor from 2014 to 2020. We assessed clinical performance, diagnostic investigations before and after (during hospitalization) surgery, tumor size and histological characteristics.Results. Among patients operated on for cardiac tumors, women predominated (74%). Among comorbidities, hypertension (79,3%), chronic kidney disease (48,3%), and obesity (25,9%) were most common. There were following clinical manifestations before surgery: shortness of breath — 47 (81%) patients, palpitations and heart rhythm disturbance — 26 (44,8%), chest pain — 25 (43,1%), chest discomfort — 28 (49,1%), edema — 6 patients (10,3%). The predominant tumor localization was the left atrial fossa ovalis area (50%). According to histological analysis, myxoma prevailed — 38 cases (86,4%). After surgery, atrial fibrillation prevalence decreased from 8 patients before surgery to 6 after surgery (p=0,034), while left atrial size decreased by 0,6 mm (95% confidence interval, 4,39-6,2 mm) (p<0,001).Conclusion. According to presented analysis over a 6-year period, cardiac neoplasms are more common in women (74,1%), while the mean age of patients is 59,7 years. Among comorbidities, hypertension prevails — 79,3%. Histological examination revealed a predominance of myxoma (86,4%). Predominant tumor location was the left atrial fossa ovalis area (50%). Surgical treatment of neoplasms was effective. So, prevalence of atrial fibrillation decreased by 25%, while left atrial size decreased by 0,6 mm. Postoperative complications and in-hospital deaths were not registered

Advances in Targeting Signal Transduction Pathways

Over the past few years, significant advances have occurred in both our understanding of the complexity of signal transduction pathways as well as the isolation of specific inhibitors which target key components in those pathways. Furthermore critical information is being accrued regarding how genetic mutations can affect the sensitivity of various types of patients to targeted therapy. Finally, genetic mechanisms responsible for the development of resistance after targeted therapy are being discovered which may allow the creation of alternative therapies to overcome resistance. This review will discuss some of the highlights over the past few years on the roles of key signaling pathways in various diseases, the targeting of signal transduction pathways and the genetic mechanisms governing sensitivity and resistance to targeted therapies

PSEUDOTUBERCULOSIS: PATHOGENETIC VALUE OF INNATE IMMUNITY CELLS

Novel data on mechanisms of innate immunity during infections with pathogenic Yersiniae are summarized in the review, that was mostly determined by complex developments regarding a unique pair of genetically related causative agents Y. pseudotuberculosis/Y. pestis. Our previous studies have revealed a morphological substrate of relative granulocyte immune deficiency that determines characteristic pathomorphologic features of pseudotuberculosis. To date, evidence has been obtained, that pathogenic for human Yersinia predominately activate protective function of innate immunity cells that is an important strategy to avoid elimination and cause the disease for the bacteria. Neutrophils (PMNs) play a fundamental role in response to infection by pathogenic Yersiniae in primary immune response and limit of primary spread of bacteria that use several mechanisms of eradication ofbacteria, e.g.: phagocytosis, oxidative stress, secretory degranulation, formation of neutrophil extracellular traps, efferocytosis. Infected PMNs can act as an intermediate host for consequent non-inflammatory infection of macrophages. Further elaboration of questions relating to primary anti-infection protection during Yersinia infections gives a key to understanding of immune pathogenesis of epidemic pseudotuberculosis (far Eastern scarlet-like fever) and yersiniosis in general

PLASMID-ASSOCIATED VIRULENCE OF YERSINIA PSEUDOTUBERCULOSIS AND INFECTIOUS PROCESS

Literature data regarding genetically determined pathogenicity factors of Y. pseudotuberculosis and associated manifestations of this infection caused by various plasmid types of the causative agent are generalized. Principal attention is given to features of cell-tissue alterations mediated by virulence plasmid pYV, as well as effects of pathogenicity of an understudied pVM82 plasmid present only in Y. pseudotuberculosis strains causing clinical-epidemic manifestation of the infections as Far East scarlet-like fever (FESLF). The data obtained on the abihty of far-eastern strains to produce YPMa super-antigen, Y. pseudotuberculosis-derivative mitogen A, probably give evidence on its key role in FESLF pathogenesis. Variability of damage of innate immunity cells and target-organs caused by various plasmid types of Y. pseudotuberculosis by virulence could determine polymorphism of clinical-morphological manifestations of this infection. In-depth understanding of dependency of immune pathogenesis mechanisms of the disease on molecular characteristics of the causative agent opens up perspectives of enhancement of diagnostics and prognosis of the severity of the course of pseudotuberculosis and yersiniosis in human in general

Möglichkeiten und Grenzen der Metallisierung textiler Flächen mittels PVD-Technologien für multifunktionale Schutztextilien

Für leichte textile Flächengebilde zum Hitze- und Kälteschutz waren durch Applikation metallischer Schichten durch thermisches Verdampfen und Magnetron-Sputtern kombinierte Shielding-Funktionen zu entwickeln. Dabei richtete sich das Augenmerk auf die Haftungsproblematik der Metallschichten ohne merklichen Verlust der Oberflächenwiderstände. Zur Verbesserung der Schichthaftung wurden die Textilien verschiedenen Vorbehandlungen, einer Gasphasenfluorierung, einer 100 nm starken Hexamethyldisiloxan-Beschichtung (HMDSO) sowie einer Behandlung mit einem Haftvermittler der Firma Bolte unterzogen. Außerdem wurden die metallisierten Materialien mit verschiedenen Zweitausrüstungen aus Polyurethan-, Vinylacetat- und zwei Polysiloxanausrüstungen nach Herstellerangaben bzw. mit einer HMDSO-Schicht behandelt. Zur Bestimmung der Haftkräfte ausgewählter Schichten dienten der Peel-Test sowie ein Martindale-Schleudertest. Parallel dazu erfolgten bis zu vier Waschprüfungen je Probe. Zur Beurteilung der elektrischen Leitfähigkeiten der Muster wurden die Oberflächenwiderstände ausgewählter Proben nach DIN EN 1149-1 mit einer Ringelektrode gemessen. Die angestrebten Haftkräfte von 8 N/15mm wurden für bedampfte und besputterte Fallschirmseide, ohne Schichtpartikel mit dem Tape vom Substrat zu lösen, erreicht und übertroffen. Die Ergebnisse im Martindale-Scheuertest zeigen, dass mit dem thermischen Bedampfen und dem Magnetron-Sputtern auf textilen Substraten Schichten mit guten Hafteigenschaften gegenüber Scheuerbeanspruchung abgeschieden werden können. Vor allem die Sputter-Beschichtung ermöglicht die Generierung sehr haftfester Schichten. Eine zusätzliche Nachbehandlung der Metallschichten mit Polymerbeschichtungen und HMDSO ist empfehlenswert. Bei den Vliesstoffen kann die Verbundfestigkeit durch eine zusätzliche Polymerbeschichtung deutlich verbessert werden. Bei der parallel zum Abriebtest erfolgten Waschbehandlung mit bis zu 4 Wäschen je Probe wurde das anvisierte Ziel, Schichthaftungen bis zu 15 Wäschen ohne merklichen Verlust der funktionellen Eigenschaften wie die Leitfähigkeit zu erzielen, nicht erreicht

Hydrogeological study of Somes-Szamos transboundary alluvial aquifer

peer reviewedSQUASH projec

THE FUNCTIONAL ACTIVITY OF INNATE IMMUNITY CELLS IN BACTERIAL INFECTION ON BACKGROUND OF THERMAL STRESS

Maintenance of thermo homeostasis under the coordinating influence of the hypothalamus is ensured by integrative interaction of various systems organisme, including the immune system. Temperature stress in infectious diseases activates the reaction of heat shock, the biochemical consequence of which is the initiation of the organism’s defense against the pathogen. Cells of innate immunity (neutrophils and macrophages) are the first line of protection against pathogenic agents and play a primary role in the development of bacterial infections. Of particular interest is the study of the duration of the effect of hyperthermia to achieve a balance between the bioenergetic costs of these cells, as well as the study of the course of the pathological process in an organism previously exposed to hige temperature. The functional status of neutrophils and macrophages, including phagocytosis, the activity of enzymes of the oxygen-dependent system: lactate dehydrogenase, cytochrome oxidase, myeloperoxidase, cellular stimulation (intracellular AMPase content) and the content of nitrogen oxide metabolites have been studied in the model of animals exposed to low and high temperatures. It has been established that under hyperthermia conditions, the change in the functional activity of cells by enzyme level is more pronounced than when exposed to animals with low temperature, especially 4 h exposure. In animals pre-exposed to heat stress, manifestations of pseudotuberculosis infection were more severe with an increase in mortality rates by 2.6 times, compared to animals infected by bacteria. These animals had a high stimulation of effector cells of inflammation in the initial periods (at 7 days) their metabolism was enhanced, which was expressed of the activity of enzymes of the oxygen-dependent system, as well as in high nitroxide-producing activity. In target organs (lung, liver, spleen) of experienced animals the severe disturbance of blood circulation in combination with significant destructive changes typical for generalized infection were showed. At dead animals on the background of marked hemorrhagic component pathological process and weak cell inflammatory response observed depletion of the immune system (delimphatization), indicating a decrease in defense reactions and the development of immunodeficiency. Thus, under conditions of heat stress (+30°С), the intensity of the reaction of innate immunity cells in terms of enzyme’s functional activity was higher than when exposed to animals of low temperature (+4°C). Under these temperature conditions, a high level of cell priming was determined, which reduced their killing potential. These data indicate the adequacy of the model used to reproduce induced secondary immunodeficiency in a congenital defense system. Moreover, in the pathogenesis of pseudotuberculosis infection against the background of prolonged action high temperature, the effects of phagocytes oxidative stress in the structural changes of immunocompetent organs were detected