Daria O. Tsymbal, Dmytro O. Minchenko, Olena Y. Luzina, Olena O. Riabovol, Serhiy V. Danilovskyi and Oleksandr H. Minchenko
Objective. The aim of the present study was to investigate the effect of adipokine NAMPT (nicotinamide phosphoribosyltransferase) silencing on the expression of genes encoding IRS1 (insulin receptor substrate 1) and some other proliferation related proteins in U87 glioma cells for evaluation of the possible significance of this adipokine in intergenic interactions.
Methods. The silencing of NAMPT mRNA was introduced by NAMPT specific siRNA. The expression level of NAMPT, IGFBP3, IRS1, HK2, PER2, CLU, BNIP3, TPD52, GADD45A, and MKI67 genes was studied in U87 glioma cells by quantitative polymerase chain reaction. Anti-visfatin antibody was used for detection of NAMPT protein by Western-blot analysis.
Results. It was shown that the silencing of NAMPT mRNA led to a strong down-regulation of NAMPT protein and significant modification of the expression of IRS1, IGFBP3, CLU, HK2, BNIP3, and MKI67 genes in glioma cells and a strong up-regulation of IGFBP3 and IRS1 and down-regulation of CLU, BNIP3, HK2, and MKI67 gene expressions. At the same time, no significant changes were detected in the expression of GADD45A, PER2, and TPD52 genes in glioma cells treated by siRNA specific to NAMPT. Furthermore, the silencing of NAMPT mRNA suppressed the glioma cell proliferation.
Conclusions. Results of this investigation demonstrated that silencing of NAMPT mRNA with corresponding down-regulation of NAMPT protein and suppression of the glioma cell proliferation affected the expression of IRS1 gene as well as many other genes encoding the proliferation related proteins. It is possible that dysregulation of most of the studied genes in glioma cells after silencing of NAMPT is reflected by a complex of intergenic interactions and that NAMPT is an important factor for genome stability and regulatory mechanisms contributing to the control of glioma cell metabolism and proliferation.
Michal Benc, Frantisek Strejcek, Matej Murin, Martin Morovic, Stanislava Martinkova, Dominika Jettmarova, Lazo Pendovski, Josef Fulka and Jozef Laurincik
An effort to improve development potential of early embryos is one of the main goals of biotechnology in the area of reproductive biology with application in veterinary or human medicine. Recent observations of the function of nucleolus or rather its forms before, during and after the fertilisation or parthenogenetic activation show the key role(s) of nucleolus in the processes of early genome activation. The nucleolus is a subnuclear structure (organelle) mainly involved in regulation of transcription and translation. This organelle has been characterized in detail by immunofluorescence, cell transfection and proteomics. This data was, however, mostly obtained in nucleoli of differentiated eukaryotic cells. Much less is known about the nucleolar structural changes and related functional processes in growing and fully grown mammalian oocytes, zygotes and early cleavage stage embryos, especially in the context of embryonic genome activation. It has been shown, that nucleoli in mammalian oocytes and early embryos have several forms and functions, which vary during the oocyte growth and embryonic development. Certain functions have not been fully described or explained, yet. The method of enucleolation, which allows to remove nucleoli from the oocytes or to exchange nucleoli between oocytes or zygotes, together with their proteomic and structural analyses brought new information about functions of nucleoli in oocytes and early cleavage-stage embryos and allowed to explain some new key roles of nucleoli during oocyte maturation and early embryonic development.
Vladimir Polaček, Dejan Vidanović, Biljana Božić, Žolt Beckei, Ivana Vučićević, Jasna Prodanov-Radulović and Sanja Aleksić-Kovacević
The most important morphological characteristic of infections caused by M. avium subsp. hominissuis (MAH) is granuloma formation. The growth of mycobacteria is in accordance with anti-bacterial effector mechanisms of the host within granuloma. The most important cytokines for „orchestrating“the host defense are interferon γ (INF-γ), tumor necrosis factor α (TNF-α) and transforming growth factor β1 (TGF-β1). Myofibroblasts that make up a peripheral layer of granuloma largely express receptors for TGF-β1. This cytokine is believed to affect the induction of myofibroblast proliferation. The aim of this paper is to point out the importance of myofibroblasts in the formation and sustainability of granuloma during natural infection of pigs with M. avium subsp. hominissuis. Examinations have been performed on the samples of Lnn. jejunales, Lnn. ileocolici and Lnn. colici of 100 pigs with a positive tuberculin skin test. The molecular method confirmed the presence of a genome M. avium subsp. hominissuis. The microscopic examination of lymph node samples stained by the routine hematoxyilin-eosin (HE) method, showed the presence of granulomatous lymphadenitis. The method of double immunohistochemical staining revealed that myofibroblasts which express TGF-β1 receptor type I (TGF-β1RI) and α smooth muscle actin (α SMA) have an important role in the morphogenesis of granulomatous lymphadenitis in pigs infected with MAH.
Ivica Gjurovski, Branko Angelovski, Toni Dovenski, Dine Mitrov and Trpe Ristoski
The aim of this study is to compare the results from the histopathology and the immunohistochemical method in the diagnostic of Porcine circovirus type 2 (PCV2) infection in pigs. The circovirus infection is a pig disease that is caused by a small, spherical, nonenveloped virus with a single stranded DNA genome which is spread throughout the pig industry worldwide. The circovirus is the etiological agent of a several pig diseases which today are thought to be the cause of the greatest economical loses in pig production. The most important of these diseases is the PMWS (post-weaning multisystemic wasting syndrome). In this article we have performed an investigation of four farms on which there had been a previous clinical diagnosis of the Post-weaning multisystemic wasting syndrome. The examination was performed on thirty pigs from these farms, from two to five months old, which had the most severe symptoms of the disease. Necropsy, histopathology and immunohistochemical diagnostic methods were performed. The most significant necropsy findings were the enlarged lymph nodes (especially the inguinal, mediasinal and the mesenteric lymph nodes). The main histopathological changes were located in the lymphatic organs presented by B and T lymphocyte depletion and increase in the number of the macrophages. PCV2 antigen and nucleic acid were detected in almost all of the examined tissues. The examination showed that the histopathological and immunohistochemical methods provide complementary results in diagnosing PCV2 in pigs.
Lozenka Ivanova, Florina Popovska-Percinic, Valentina Slavevska-Stamenkovic, Maja Jordanova and Katerina Rebok
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Nikola Adamov, Dine Mitrov, Igor Esmerov and Peter Dovc
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Fatemeh Khatami, Mahsa Mohammadamoli and Seyed Mohammad Tavangar
Pheochromocytomas and paragangliomas (PPGLs) are tumors arising from the adrenal medulla and sympathetic/parasympathetic paraganglia, respectively. According to Th e Cancer Genome Atlas (TCGA), approximately 40% of PPGLs are due to germ line mutations in one of 16 susceptibility genes, and a further 30% are due to somatic alterations in at least seven main genes (VHL, EPAS1, CSDE1, MAX, HRAS, NF1, RET, and possibly KIF1B). Th e diagnosis of malignant PPGL was straight forward in most cases as it was defined as presence of PPGL in non-chromaffin tissues. Accordingly, there is an extreme need for new diagnostic marker(s) to identify tumors with malignant prospective. Th e aim of this study was to review all suggested genetic and epigenetic alterations that are remarkably different between benign and malignant PPGLs. It seems that more than two genetic mutation clusters in PPGLs and other genetic and methylation biomarkers could be targeted for malignancy discrimination in different studies.