We are experiencing rapid progress in all types of imaging techniques used in the detection of various numbers and types of mutation. In situ hybridization (ISH) is the primary technique for the discovery of mutation agents, which are presented in a variety of cells. The ability of DNA to complementary bind is one of the main principles in every method used in ISH. From the first use of in situ techniques, scientists paid attention to the improvement of the probe design and detection, to enhance the fluorescent signal intensity and inhibition of cross-hybrid presence. This article discusses the individual types and modifications, and is focused on explaining the principles and limitations of ISH division on different types of probes. The article describes a design of probes for individual types of in situ hybridization (ISH), as well as the gradual combination of several laboratory procedures to achieve the highest possible sensitivity and to prevent undesirable events accompanying hybridization. The article also informs about applications of the methodology, in practice and in research, to detect cell to cell communication and principles of gene silencing, process of oncogenesis, and many other unknown processes taking place in organisms at the DNA/RNA level.
AA genotype of A 1166C polymorphism in the ATR1 gene may be associated with hypotension and decline in sympathetic tone during HUT. Its role in genetic predisposition to vasovagal syncope cannot be excluded.
The innate response of melanocytes to exogenous or endogenous stress stimuli like extreme pH and temperature, metabolite and oxygen deficiency or a high UV dose initiates a cellular stress response. This process activates adaptive processes to minimize the negative impact of the stressor on the pigment cell. Under physiological conditions, a non-cancer cell is directed to apoptosis if the stressor persists. However, malignant melanoma cells will survive persistent stress thanks to distinct "cancerous" signaling pathways (e.g. MEK) and transcription factors that regulate the expression of so-called "survival genes" (e.g. HIF, MITF). In this survival response of cancer cells, MEK pathway directs melanoma cells to deregulate mitochondrial metabolism, to accumulate reduced species (NADH), and to centralize metabolism in the cytosol. The aim of this work was to study the effect of gene silencing in malignant melanoma A375 cells on metabolic processes in cytosol and mitochondria. Gene silencing of HIF-1α, and miR-210 in normoxia and pseudohypoxia, and analysis of its effect on MITF-M, and PDHA1 expression. Detection of cytosolic NADH by Peredox-mCherry Assay. Detection of OCR, and ECAR using Seahorse XF96. Measurement of produced O2•− with MitoTracker Red CMXRos. 1H NMR analysis of metabolites present in cell suspension, and medium. By gene silencing of HIF-1α and miR-210 the expression of PDHA1 was upregulated while that of MITF-M was downregulated, yielding acceleration of mitochondrial respiratory activity and thus elimination of ROS. Hence, we detected a significantly reduced A375 cell viability, an increase in alanine, inositol, nucleotides, and other metabolites that together define apoptosis. Based on the results of measurements of mitochondrial resipiratory activity, ROS production, and changes in the metabolites obtained in cells under the observed conditions, we concluded that silencing of HIF-1α and miR-210 yields apoptosis and, ultimately, apoptotic cell death in A375 melanoma cells.
Nováková J., D. Daňová, k. Strišková, R. Hromada, H. Mičková, M. Rabišková: Zinc and Cadmium Toxicity Using a Biotest with Artemia franciscana. acta vet. Brno 2007, 76: 635-642. of the various toxic elements heavy metals, particularly cadmium, lead, mercury and zinc, occur frequently in the environment due to their relatively high industrial use. While the toxicity of individual substances is usually well known, information about their mutual interactions is relatively scarce. in animal experiments the prevailing trend is to substitute higher vertebrates with biotests of the 2 nd generation. our experiment focused on observation of the effect of combinations of ZnSo 4 .7H 2 o and CdCl 2 .2H 2 o on lethality to Artemia franciscana. The aim of the study was to observe the synergistic or antagonistic effects of these two metals.depending on concentration, cadmium may increase or decrease the toxicity of zinc. at higher concentrations of CdCl 2 .2H 2 o exceeding 100 mg·l -1 one can observe obvious synergistic toxic effects of both the substances. our observations allowed us to conclude that the use of optimum, relatively low concentrations of cadmium (up to 50 mg·l -1 CdCl 2 .2H 2 o) results in a significant decrease in lethality to Artemia franciscana caused by ZnSo 4 .7H 2 o at concentrations of 50, 100 a 250 mg·l -1 .
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