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Introduction. Non-invasive methods of research of the human organism clinical conditions have been widely used in medicine as a diagnostic and prognostic test. Since the protein content of human urine is closely related to the peculiarity of the functioning of biological systems, its qualitative and quantitative characteristics are promising markers for control of various processes in organism. An important source of protein markers is the results of detailed proteomics (two-dimensional electrophoresis of proteins followed by mass spectrometry) of human urine, depending on the disease. Recently, the protein of SRC8 (Sarc family protein kinase substrate or cortactin) was detected among 12 new proteins in urine protein analyzes in breast cancer patients. The aim of the study. To investigate the spectrum of cortactin isoforms in the urine of patients with liver cirrhosis (LC) and their diagnostic value as a potential molecular marker of its severity. Materials and methods. In a randomized pre-stratified method, 18 patients (women – 41.7 %, males – 58.3 %) of 54.8 ± 2.5 years old who were treated at Lviv Regional Hepatological Center based on the department of Internal Medicine №1 of Danylo Halytsky Lviv National Medical University and Gastroenterological Department of the Communal Nonprofit Enterprise of the Lviv Regional Council ″Lviv Regional Clinical Hospital″. The experimental group (EG) was formed of 12 patients (100.0 %) with LC of different etiology (6 people (50.0 %) – toxic-alimentary, 1 (8.3 %) – C-viral, 2 (16.7 %) – primary biliary, 2 (16.7 %) – toxic-alimentary + C-viral, 1 (8.3 %) – autoimmune + toxic-alimentary. The control group (CG) was formed of 10 practically healthy persons of the corresponding age and gender. Patients were provided with a comprehensive clinical-laboratory and instrumental examination of all organs and systems in accordance with the requirements of modern medicine. In addition, in all of the examined persons 10.0 ml of urine morning portion was taken, it was prepared according to the procedure, and subsequently the proteins with acetone were precipitated in a ratio of 1:6 and subjected to SDS-electrophoresis followed by Western blot analysis using commercial monospecific anti-cortactin antibodies. Statistical processing of information was carried out using programs Microsoft Office Excel 2010 with the estimation of the authenticity of the revealed changes using the Student’s t-criterion (W. S. Gosett) (the difference was considered significant for p < 0.05) were done. Diagnostic sensitivity of the certain protein was determined by the diagnostic specificity, prognostic significance of positive results, prognostic significance of negative results, and diagnostic effectiveness of the parameter. Results. During the study of the spectrum of immunologically reactive proteins in urine specimens of 12 patients with LC it was found that 9 of 12 patients had polypeptide with a molecular weight of 64 kDa, 4 subjects – 52 kDa protein, 8 – 24 kDa protein. It also has been found that immunoglobulin-related cortactin polypeptides with a molecular weight of 98, 64 and 24 kDa were present in the CG – 9 out of 10 urine specimens of healthy people contained protein with molecular weight of 64 kDa, one – 24 kDa, one – 98 kDa. According to the comparative analysis of the results obtained, it can be argued that 24 kDa protein found in 8 patients with LC was observed only in one of 10 urine specimens of the controls; 64 kDa polypeptide – in 9 persons of EG and 9 controls; 98 kDa – in nobody from the EG and only in the urine of one patient of the CG; 52 kDa protein – in 4 persons of EG and in nobody from the controls. 52 kDa protein, found in individuals of the EG, was not detected in the urine of any patient from the CG. After analyzing the information on 4 patients in whose urine 52 kDa protein was identified, it was found that in all LC was at the stage of decompensation (class C according to the criteria of C. H. G. Child – R. N. Pugh), in connection with which it can be assumed that the appearance of this protein in urine may be associated with the LC severity increase. 24 kDa protein was significantly more commonly found in the EG (8 persons – 6 patients had LC at the stage of decompensation, class C according to the criteria of C. H. G. Child – R. N. Pugh, in two – at the stage of subcompensation, class B according to the criteria of C. G. Child – R. N. Pugh) than in the CG (1 person) (p = 0.0039). Conclusions. Investigation of protein content in urine using western blot analysis using commercial antibodies to human cortactin allowed the identification of various molecular forms of peptides that differ in patients with liver cirrhosis of different etiology and healthy persons: the protein with the molecular weight of 52 kDa was present only in the urine of the experimental group patients, 24 kDa polopeptide was significantly (p = 0.0039) more likely common in the experimental than in the control group. The increase of the detection rate of 24.0 kDa proteins and the appearance of 52.0 kDa proteins in the urine may be associated with the appearance of the decompensated stage of liver cirrhosis. However, the assessment of their diagnostic value needs the further research.

Rationale: Dysferlin myopathy is an autosomal recessive hereditary muscular dystrophy due to deficiency of dysferlin caused by alteration of the DYSF gene; Limb-girdle muscular dystrophy type 2B (LGMD2B) is the most common in Its clinical phenotypes. However, LGMD2B is rarely seen in clinical cases and may initially present as weakness of proximalpelvis muscles and muscles in the posterior compartments of thighs,which will then cause difficulty in running and limping during walking. Laboratory tests at an early stage of the disease often indicate an increased level of serum creatine kinase (CK). Moreover, polymyositis (PM) is manifested as symmetrical proximal muscle weakness of the four limbs, accompanied by an increased level of serum CK. Thus, both are very difficult to identify in clinical practice. Patient concerns: A 25-year-old woman was admitted to our department as the limb weakness progressively worsened. She began to experience proximal muscle weakness of both lower limbs without obvious inducement, which markedly increased when she climbed the stairs or stood up after squatting. Then her symptoms worsened, with difficulty in proximal and distal lifting of the lower extremities. Diagnoses: Through combined immunohistochemistry and Western-blot analysis, The patient was diagnosed with LGMD2B. Interventions: There were symptomatic treatments such as coenzyme Q10. Outcomes: After symptomatic treatments, the patient's symptoms were obviously relieved, and the CK level decreased. Lessons: Through this case, we found that combined application of immunohistochemistry and Western-blot analysis is helpful in early diagnosis of LGMD2B, and a new site of frame-shift mutation in the patient's DYSF gene was found.

WAG/Rij rat strain is inherited model of human childhood absence epilepsy. Genetic mechanisms of seizures are unknown but it was shown that reticular thalamic nucleus (RTn) and somatosensory cortex (’cortical focus’) are involved in appearing of spike-wave discharges (SWD) origin. T-type calcium channels are long suspected players in development of SWD in thalamocortical loop; Kir 4.1 is essential channel for normal physiology of cortical astrocytes involved in the glutamate uptake. We examined mRNA expression level of Kir 4.1 in somatosensory ’cortical focus’ and three T-channels alpha1-subunits in both ’cortical focus’ and RTn of WAG/Rij strain compared to control Wistar rats by means of RT-qPCR. We found CACNA1I>>CACNA1G>CACNA1H in RTn of both strains. In ’cortical focus’ tissue mRNA quantity was CACNA1I>CACNA1G>>CACNA1H for Wistar and CACNA1I=CACNA1G>>CACNA1H for WAG/Rij strain. CACNA1G mRNA is slightly upregulated in RTn and 3-fold in ’cortical focus’of WAG/Rij. Level of CACNA1H is also higher in both brain regions in WAG/Rij rats. CACNA1I showed insignificant decreasing in both studied tissues of WAG/Rij strain. Level of Cav 3.1 protein was increased in ’cortical focus’ tissue of WAG/Rij. We also demonstrated stable level of Kir 4.1 mRNA expression in ’cortical focus’. Our results may suggest CACNA1G involvement in SWD generation in somatosensory cortex while role of other T-type calcium channel subunits and Kir 4.1 is less possible.

Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC. © 2017, Springer Science+Business Media New York.

Secretory immunoglobulin A (sIgA) contains glycoconjugates moieties which can be involved in humoral immunity providing antimicrobial defense. The aim of present work was to characterize carbohydrate moieties of sIgA isolated from human colostrum via their interaction with specific lectins. Preparations of sIgA were obtained from 23 colostrums samples of parturient women by 3-fold precipitation with in ammonium sulphate 50% saturation. In the study a panel of 44 lectins of known carbohydrate specificity was used immobilized onto nitrocellulose membrane. The panel was incu­bated with sIgА preparations and lectin-antibody complexes were detected by protein-A conjugated with horseradish peroxidase. Western-blot analysis was applied to cha­racterize glycosylated polypeptides of sIgA isolated from colostrum of parturient women. sIgA from the colostrum of clinically healthy parturient women contained mainly carbohydrate moieties consisting of N-acetyl-D-glucosamine, sialic acid and D-mannose. Substantial differences in interaction of carbohydrate determinants of slgA isolated from colostrum of individual parturient women with the lectines were found. It is proposed that such characteristics can define sensitivity of mother and child to the action of pathogenic microflora.

Мета: Визначити, як експресується MGMT ген на рівні білка у клітинах інтактного паро- донта та при запаленні, спричиненому різними бактеріальними збудниками. Методи: Об’єктом дослідження були тканини пародонта 30 двомісячних щурів, яких розділили на 5 груп, по 6 тварин у кожній. Тваринам першої групи в ясенну борозну викладали нитки з чистою культурою Candida albicans, другої групи — Staphylococcus haemolyticus, третьої — Staphylococcus aureus. Для контролю метилування тваринам четвертої групи одноразово вводили MNNG (Methylnitronitrosoguanidine). Тварини п’ятої групи були ін- тактними, зі здоровим пародонтом і склали контролльну групу. Наявність MGMT у ліза- тах клітин пародонта виявили за допомогою Вестерн-блот аналізу. Контроль нанесення проводили методом денситометрії мембрани за допомогою програми OriginPro 8.5, статистичний аналіз результатів — за критерієм Стьюдента. Результати: Аналіз зразків пародонта показав статистично значимі залежності вибірок за критерієм Стьюдента для Candida albicans: t=2,566 (P≤0.01), для Staphylococcus aureus: t=5,059 (P≤0,003), для Staphylococcus haemolyticus: t=4,332 при (P≤0,007). Це доводить існування залежності рівня експресії MGMT на рівні білка від збудника при запаленні пародонта. При по- рівнянні експресії MGMT у контрольній групі та сумарного рівня у піддослідних щурів виявили достовірну різницю t=2,432 (P≤0.02). Висновки: Довели можливість неспеци- фічного індукованого підвищення експресії MGMT бактеріальними продуктами при модельованому пародонтиті. Виявили різну залежність рівня експресії гена MGMT при запаленні пародонта, спричиненому досліджуваними бактеріями. Встановили імовір- ність кумулятивної дії ендо- та екзотоксинів бактерій на експресію гена MGMT. У по- дальшому необхідно дослідити пошкодження генів DNA microarray методом у хворих на запальні захворювання пародонта. При цитуванні документа, використовуйте посилання http://essuir.sumdu.edu.ua/handle/123456789/32914 Purpose: Analyze the expression of MGMT in intact cells and marginal periodontitis. Methods: periodontal tissues of 30 rats two months old, which were divided into 5 groups (6 on each). The animals of the first group has injected strands in gingival sulcus with Candida albicans, the animals of the second group - Staphylococcus haemolyticus, third groups of Staphylococcus aureus. The animals of the fourth group once has injected strands in gingival sulcus with MNNG (Methylnitronitrosoguanidine) (for the control of methylation). Animals fifth group has a healthy periodontium and served as a control. The presence of MGMT in periodontal we identify by Western blot analysis. Densitometry of membrane has performed by using the program OriginPro 8.5. Statistical analysis of densitometry is carried out by Student's test for independent samples. Results: The analysis of periodontal tissues samples experimental rats showed a statistically significant sample depending on the criterion Student for Candida albicans t=2,566 (P≤0.01), for Staphylococcus aureus t=5,059 (P≤0,003), for Staphylococcus haemolyticus t= 4,332 at (P≤0,007). This shows the existence dependent expression levels of MGMT protein with pathogen in periodontal inflammation. Compare MGMT expression in control animals and total level expression of MGMT in experimental rats we found t=2,432 (P≤0.02). Conclusions: The modeled presence of the marginal periodontitis is induced MGMT expression by bacterial products. We found a different dependence of MGMT gene expression by different bacterial pathogen. The established cumulative possibility of endo-and exotoxins of bacteria on the expression of MGMT. При цитировании документа, используйте ссылку http://essuir.sumdu.edu.ua/handle/123456789/32914

Insulin is the principal regulatory hormone involved in the tight regulation of fuel metabolism. In response to blood glucose levels, it is secreted by the beta cells of the pancreas and exerts its effects by binding to cell surface receptors that are present on virtually all cell types and tissues. The insulin receptor (IR) is a transmembrane tyrosine kinase that becomes activated upon ligand binding. Consequently, the receptor and its downstream substrates become tyrosine phosphorylated. This activates a series of intracellular signaling cascades which coordinately initiate the appropriate biological response like insertation of glucose transporter 4 (GLUT4) in to the plasma membrane. One important mechanism by which insulin signaling is regulated involves the protein tyrosine phosphatases (PTPs), which may either act on the insulin receptor itself and/or its substrates. In our work we have isolated and purified antibodies for the detection of betha-subunit of IRKD. Antibodies are used to confirm the expression of IRKD recombinant protein using Baculovirus expression system in Sf9 cells. Two peptides have been synthesized whose amino acid sequences are equal to the different sequences of the C-terminal end of the  -subunit of the IRKD. The peptides have been used as antigens for immunization of rabbits. During the immunization time of 6 months rabbits have produced sequence specific polyclonal antibodies. Antibody production in the rabbit serum has been detected by ELISA test. Produced antibodies have been isolated from the rabbit’ s immune serum and purified by following methods: precipitation by ammonium sulfate and affinity chromatography on protein A and petide-sepharose columns. The specificity of produced antibodies were determinated by Elisa-test and Western-blot analysis.

At least three types of mRNA of the catalytic subunit of Na,K-ATPase namely alpha-,alpha+- and alpha 3-isoforms are identified in different tissues. Only two of them alpha and alpha+ have well known structural and catalytic properties. Here we present immunochemical data indicating that the alpha 3 protein really exists in pig and human kidney, and human brain. Crude membrane fractions and purified membrane-bound Na,K-ATPases were immunoblotted with alpha 3-specific antibodies raised against the synthetic peptide corresponding to the unique sequence of this isoform. The mature alpha 3-subunit is shown to include the sequence GDKKDDKSSPK followed by the initiating methionine residue. Nephron collecting tubules are proposed to specifically contain Na,K-ATPase alpha 3-isoform.