Reactions In Human Cells Research Articles

High-dose copper activates p53-independent apoptosis through the induction of nucleolar stress in human cell lines.

Copper is an essential micronutrient involved in many redox reactions in human cells. However, a high concentration of copper, intake from the environment or abnormal accumulation within cells because of genetic mutation, leads to cell toxicity. This is attributable to oxidative damage, altered gene expression, and functional impairment of the mitochondria. Copper stress also alters the morphology of the nucleolus, but the process has not been fully elucidated. In this study, cells were treated with copper sulfate at 3-9ppm and examined if a high dose of copper would block ribosome biogenesis. With the incorrect distribution of nucleolar proteins nucleophosmin and fibrillarin to the nucleoplasm, ribosomal RNA (rRNA) processing was impaired; 34S rRNA from an abnormal A2 cut increased, and downstream pre-rRNAs decreased. The under-accumulation of 60S subunits was detected using sucrose gradients. From transcriptome analysis, ribosome synthesis-related genes were misregulated. Blockage in ribosome synthesis under copper-treatment induced nucleolar stress and triggered p53-independent apoptosis pathways. Thus, nucleolar stress is one cause of cell death under copper exposure.

ВЛИЯНИЕ РАСТИТЕЛЬНЫХ ЭКСТРАКТОВ НА УРОВЕНЬ СВОБОДНО РАДИКАЛЬНЫХ ПРОЦЕССОВ В КЛЕТКАХ ЧЕЛОВЕКА

We evaluated the effects of extracts from the pomegranate (Punica granatum), vine (Vitis vinifera) seeds and garlic (Allium sativum) on free-radical reactions in human cells in relation to SNPs in the SOD2and СAT genes. The object was human peripheral leukocytes cultured in the presence of extracts from pomegranate (1.2; 2.4 %), garlic (0.5; 1.2 %) and vine seeds (1.2; 2.4 %). Intensity of free-radical reactions was assessed by a fast flash and flashlight sum of the luminol-dependent chemiluminescence (LDCL). Alleles of SOD2and СAT were analyzed using allele-specific PCR. The results showed that the pomegranate extract decreases LDCL intensity reliably in comparison with the control. Extracts of the garlic (1.2 %) and vine seeds (1.2 %) increase LDCL intensity. Correlation was established between the genotype determined by polymorphism of SOD2 gene Ala16Val (rs4880) and fast flash intensity during human cell cultivation with the extract of vine seeds (1.2 %).

Dietary Advanced Glycation Endproducts Decrease Glucocorticoid Sensitivity In Vitro.

Glucocorticoids are very effective anti-inflammatory drugs and widely used for inflammatory bowel disease (IBD) patients. However, approximately 20% of IBD patients do not respond to glucocorticoids and the reason for this is largely unknown. Dietary advanced glycation endproducts (AGEs) are formed via the Maillard reaction during the thermal processing of food products and can induce a pro-inflammatory reaction in human cells. To investigate whether this pro-inflammatory response could be mitigated by glucocorticoids, human macrophage-like cells were exposed to both LPS and AGEs to induce interleukin-8 (IL8) secretion. This pro-inflammatory response was then modulated by adding pharmacological compounds interfering in different steps of the anti-inflammatory mechanism of glucocorticoids: rapamycin, quercetin, and theophylline. Additionally, intracellular reactive oxygen species (ROS) were measured and the glucocorticoid receptor phosphorylation state was assessed. The results show that AGEs induced glucocorticoid resistance, which could be mitigated by quercetin and rapamycin. No change in the phosphorylation state of the glucocorticoid receptor was observed. Additionally, intracellular ROS formation was induced by AGEs, which was mitigated by quercetin. This suggests that AGE-induced ROS is an underlying mechanism to AGE-induced glucocorticoid resistance. This study shows for the first time the phenomenon of dietary AGE-induced glucocorticoid resistance due to the formation of ROS. Our findings indicate that food products with a high inflammatory potential can induce glucocorticoid resistance; these results may be of great importance to IBD patients suffering from glucocorticoid resistance.

A mathematical analysis of Prx2-STAT3 disulfide exchange rate constants for a bimolecular reaction mechanism.

Appreciation of peroxiredoxins as the major regulators of H2O2 concentrations in human cells has led to a new understanding of redox signaling. In addition to their status as the primary reducers of H2O2 to water, the oxidized peroxiredoxin byproduct of this reaction has recently been shown capable of participation in H2O2-mediated signaling pathways through disulfide exchange reactions with the transcription factor STAT3. The dynamics of peroxidase-transcription factor disulfide exchange reactions have not yet been considered in detail with respect to how these reactions fit into the larger network of competing reactions in human cells. In this study, we used a kinetic model of oxidation and reduction reactions related to H2O2 metabolism in the cytosol of human cells to study the dynamics of peroxiredoxin-2 mediated oxidation of the redox-regulated transcription factor STAT3. In combination with previously reported experimental data, the model was used to estimate the rate coefficient of a biomolecular reaction between Prx2 and STAT3 for two sets of assumptions that constitute lower and upper bound cases. Using these estimates, we calculated the relative rates of the reaction of oxidized peroxiredoxin-2 and STAT3 and other competing reactions in the cytosol. These calculations revealed that peroxiredoxin-2-mediated oxidation of STAT3 likely occurs at a much slower rate than competing reactions in the cytosol. This analysis suggests the existence of more complex mechanisms, potentially involving currently unknown protein-protein recognition partners, which facilitate disulfide exchange reactions between peroxiredoxin-2 and STAT3.

Comparison of eluted monomer from different indirect and direct resin composites

Indirect resin composites used in indirect restorations have been used as alternative methods to avoid the negative effects of a direct application. The composition and structure of these are similar to a direct composite. Several authors have discussed the elution monomer from resin composites because it is a crucial factor in optimizing the physical properties and biocompatibility of resin composites. The aim of this study was to compare the elution of monomers from different resin composites using high-performance liquid chromatography (HPLC). Indirect composites including Signum, Gradia, and Solidex, and direct composite Filtek Ultimate were used. The samples (2 x 5 mm) were prepared and polymerized for 20 seconds with a light-emitting diode unit. A secondary curing was then applied to the indirect resin composites. After fabrication, each sample was immediately immersed in a 75 wt% ethanol/water solution and analyzed by HPLC. The obtained data were analyzed with one-way ANOVA and Tukey HSD (p < 0.05). Residual monomers were eluted from indirect and direct resin composites and the amount of eluted monomers increased over time. The highest mean concentrations of residual Bis-GMA, UDMA, TEGDMA, and HEMA were detected with the Filtek Ultimate (2,342 µM), Solidex (7,908 µM), Signum (2.8 µM), and Filtek Ultimate (9.7 µM), respectively. The highest amount of eluted monomer concentrations detected was viewed as critical for toxic reactions in human cells.

Target-specific variants of Flp recombinase mediate genome engineering reactions in mammalian cells.

Genome engineering relies on DNA-modifying enzymes that are able to locate a DNA sequence of interest and initiate a desired genome rearrangement. Currently, the field predominantly utilizes site-specific DNA nucleases that depend on the host DNA repair machinery to complete a genome modification task. We show here that genome engineering approaches that employ target-specific variants of the self-sufficient, versatile site-specific DNA recombinase Flp can be developed into promising alternatives. We demonstrate that the Flp variant evolved to recombine an FRT-like sequence, FL-IL10A, which is located upstream of the human interleukin-10 gene, and can target this sequence in the model setting of Chinese hamster ovary and human embryonic kidney 293 cells. This target-specific Flp variant is able to perform the integration reaction and, when paired with another recombinase, the dual recombinase-mediated cassette exchange reaction. The efficiency of the integration reaction in human cells can be enhanced by 'humanizing' the Flp variant gene and by adding the nuclear localization sequence to the recombinase.

Homologous DNA Pairing by Human Recombination Factors Rad51 and Rad54

Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. We show an ability of hRad54 to promote transient separation of the strands in duplex DNA via its ATP hydrolysis-driven DNA supercoiling function. The ATPase, DNA supercoiling, and DNA strand opening activities of hRad54 are greatly stimulated through an interaction with hRad51. Importantly, we demonstrate that hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. Our results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells.