Analysis of Plasma Membrane Integrity by Fluorescent Detection of Tl+ Uptake

The exclusion of polar dyes by healthy cells is widely employed as a simple and reliable test for cell membrane integrity. However, commonly used dyes (propidium, Yo-Pro-1, trypan blue) cannot detect membrane defects which are smaller than the dye molecule itself, such as nanopores that form by exposure to ultrashort electric pulses (USEPs). Instead, here we demonstrate that opening of nanopores can be efficiently detected and studied by fluorescent measurement of Tl⁺ uptake. Various mammalian cells (CHO, GH3, NG108), loaded with a Tl⁺-sensitive fluorophore FluxOR™ and subjected to USEPs in a Tl⁺-containing bath buffer, displayed an immediate (within <100 ms), dose-dependent surge of fluorescence. In all tested cell lines, the threshold for membrane permeabilization to Tl⁺ by 600-ns USEP was at 1–2 kV/cm, and the rate of Tl⁺ uptake increased linearly with increasing the electric field. The lack of concurrent entry of larger dye molecules suggested that the size of nanopores is less than 1–1.5 nm. Tested ion channel inhibitors as well as removal of the extracellular Ca²⁺ did not block the USEP effect. Addition of a Tl⁺-containing buffer within less than 10 min after USEP also caused a fluorescence surge, which confirms the minutes-long lifetime of nanopores. Overall, the technique of fluorescent detection of Tl⁺ uptake proved highly effective, noninvasive and sensitive for visualization and analysis of membrane defects which are too small for conventional dye uptake detection methods.     

Transforming growth factor-beta-activated kinase-1 (TAK1), a MAP3K, interacts with Smad proteins and interferes with osteogenesis in murine mesenchymal progenitors

TAK1 (transforming growth factor-beta-activated kinase-1), a MAP3K with considerable sequence similarity to Raf-1 and MEKK-1, has been identified as a transforming growth factor-beta/bone morphogenetic protein (BMP)-activated cytosolic component of the MAPK pathways. In this investigation, the molecular interactions between TAK1 and Smad proteins were characterized as well as their influence on BMP-mediated mesenchymal cell differentiation along the osteogenic/chondrogenic pathway. In co-immunoprecipitations we found an interaction of TAK1 with all Smads tested, R-Smads Smads1-5, the co-Smad Smad4, and the inhibitory Smads (I-Smad6 and I-Smad7). Smad interaction with TAK1 takes place through their MH2 domain. This interaction is dependent on the presence of an active kinase domain in TAK1. TAK1 dramatically interferes with R-Smad transactivation in reporter assays and affects subcellular distribution of Smad proteins. Activated TAK1 also interferes with BMP-dependent osteogenic development in murine mesenchymal progenitor cells (C3H10T 1/2). A potential TAK1-mediated apoptosis process could be excluded for these cells. Both synergistic and interfering influences of TAK1 on BMP-mediated Smad-signaling have been reported previously. We suggest that TAK1 is a factor that is involved in the fine-tuning of BMP effects during osteogenic development.     

Beneficial Effect of Fenofibrate and Silymarin on Hepatic Steatosis and Gene Expression of Lipogenic and Cytochrome P450 Enzymes in Non-Obese Hereditary Hypertriglyceridemic Rats

The efficacy of fenofibrate in the treatment of hepatic steatosis has not been clearly demonstrated. In this study, we investigated the effects of fenofibrate and silymarin, administered as monotherapy and in combination to existing hepatic steatosis in a unique strain of hereditary hypertriglyceridemic rats (HHTg), a non-obese model of metabolic syndrome. HHTg rats were fed a standard diet without or with fenofibrate (100 mg/kg b.wt./day) or with silymarin (1%) or with a combination of fenofibrate with silymarin for four weeks. Fenofibrate alone and in combination with silymarin decreased serum and liver triglycerides and cholesterol and increased HDL cholesterol. These effects were associated with the decreased gene expression of enzymes involved in lipid synthesis and transport, while enzymes of lipid conversion were upregulated. The combination treatment had a beneficial effect on the gene expression of hepatic cytochrome P450 (CYP) enzymes. The expression of the CYP2E1 enzyme, which is source of hepatic reactive oxygen species, was reduced. In addition, fenofibrate-induced increased CYP4A1 expression was decreased, suggesting a reduction in the pro-inflammatory effects of fenofibrate. These results show high efficacy and mechanisms of action of the combination of fenofibrate with silymarin in treating hepatic steatosis and indicate the possibility of protection against disorders in which oxidative stress and inflammation are involved.     

Physiological comparison of cells with high and low alcohol dehydrogenase activities in bacterial populations consuming ethanol

Measuring the physiological heterogeneity of natural and industrial microbial populations is essential to studying, modelling and monitoring of microbial populations. It was discovered that populations of Escherichia coli and Bacillus megaterium growing in medium with ethanol as an external source of energy have two actively respiring but physiologically different subpopulations. Cells of one subpopulation have negligibly low alcohol dehydrogenase (ADH) activity (ADH-L cells) and cells of the other have high ADH activity (ADH-H cells). The subpopulation of ADH-H bacterial cells was measured using 10 min incubations of cells in a 1% solution of allyl alcohol for fast selective killing of cells with high activity of ADH and flow cytometry detection of dead cells after this incubation. The content of ADH-H cells during exponential phase of batch culture varied from 9 % to 90 % and lowered to zero for a few hours during starvation of the population. ADH-L cells are actively respiring cells and not depolarized cells. The simultaneous presence of ADH-L and ADH-H cells growing in the medium with ethanol can be explained by the fact that ADH-H cells oxidize actively external ethanol whereas ADH-L cells oxidize only intracellular storage carbohydrates. The method for enumeration of cells with high ADH activity can be used to monitor the heterogeneity of bacterial populations consuming ethanol as a sole source of carbon and energy.     

Structure of the extremely slow GTPase Rab6A in the GTP bound form at 1.8 Å resolution

Rab/Ypt GTPases represent a>60 member large family of membrane traffic regulators in eukaryotic cells. Members of this group display intrinsic GTPase activity varying over two orders of magnitude. Here, we show that Rab6A represents the RabGTPase with the slowest spontaneous GTPase activity yet measured (5x10(-6)s(-1)). Due to the very low intrinsic hydrolysis rate we were able to crystallise and solve the structure of the Rab6A:GTP complex to 1.82A resolution. Analysis of the structure suggests that low catalytic activity of the Rab6A might be due to high flexibility of the Switch II region and a low degree of constraint of critically important for catalysis Gln 72.     

PPARGC1A gene polymorphism is associated with exercise-induced fat loss

Molecular Biology Reports - Obesity is a widespread problem within modern society, serving to increase the risk of cardiovascular, metabolic, and neurodegenerative disorders. Peroxisome...     

Novel N‐Cycloalkylcarbonyl‐N′‐arylthioureas: Synthesis,Design, Antifungal Activity and Gene Toxicity

A synthesis method of novel N‐cycloalkylcarbonyl‐N′‐arylthioureas was developed. It consists of sequential addition of equimolecular amounts of ammonium isothiocyanate and substituted anilines to cycloalkylcarbonyl chlorides. The identity and purity of products were confirmed by LC/MS spectra, their structure by elemental analysis, IR and ¹H‐NMR spectra. Preliminary antimicrobial screening for standard microorganisms and molecular docking allowed to select several structures for antifungal and genetic toxicity studies. Conducted in vitro screening of 9 compounds for antifungal potential against 11 phytopathogenic fungi and three Phytophthora strains revealed that two N‐(arylcarbamothioyl) cyclopropanecarboxamides at a concentration of 50 μg/ml exhibited activities comparable to the standard antifungal agent ‘Cyproconazole’. Analysis of mutagenicity of novel thioureas using the Salmonella reverse mutagenicity assay (‘Ames Test’) showed a low gene‐toxicity profile.     

Recombinant DNA production of spider silk proteins

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks.     

Immortalized cells and one oncogene in malignant transformation: old insights on new explanation

Background

Nearly thirty years ago, it was first shown that malignant transformation with single oncogene necessarily requires the immortal state of the cell. From that time this thesis for the cells of human origin was not disproved. The basic point which we want to focus on by this short communication is the correct interpretation of the results obtained on the widely used human embryonic kidney 293 (HEK293) cells.

Results

Intensive literature analysis revealed an increasing number of recent studies discovering new oncogenes with non-overlapping functions. Since the 1970s, dozens of oncogenes have been identified in human cancer. Cultured cell lines are often used as model systems in these experiments. In some investigations the results obtained on such cells are interpreted by the authors as a malignant transformation of normal animal or even normal human cells (as for example with HEK293 cells). However, when a cell line gains the ability to undergo continuous cell division, the cells are not normal any more, they are immortalized cells. Nevertheless, the authors consider these cells as normal human ones, what is basically incorrect. Moreover, it was early demonstrated that the widely used human embryonic kidney 293 (HEK293) cells have a relationship to neurons.

Conclusions

Thus, the experiments with established cell lines reinforce the notion that immortality is an essential requirement for malignant transformation that cooperates with other oncogenic changes to program the neoplastic state and substances under such investigation should be interpreted as factors which do not malignantly transform normal cells alone, but possess the ability to enhance the tumorigenic potential of already immortalized cells.