A critical role of tropomyosins in TGF-beta regulation of the actin cytoskeleton and cell motility in epithelial cells

We have investigated transforming growth factor beta (TGF-beta)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-beta via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, alpha-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-beta induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-beta induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-beta induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-beta induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-beta control of cell motility, and the loss of this TGF-beta response is a critical step in the acquisition of metastatic phenotype by tumor cells.     

Influence of the activation of the immune system cells on the parameters of lipid metabolism in macrophages

The influence of tumor necrosis factor a (TNF-alpha) and media, conditioned by activated macrophages and lymphocytes and containing a complex of biologically active compounds (including cytokines), on the parameters of lipid metabolism in macrophages was studied. The addition of recombinant TNF-alpha and immunocompetent cell-conditioned media to mouse peritoneal macrophages culture stimulated labelled oleate incorporation into cholesterol esters and triglycerides, as well as labelled glycerine incorporation into cholesterol esters, but inhibited labelled cholesterol incorporation into cholesterol esters. One of the mechanisms of the influence of activated immunocompetent cells on cholesterol metabolism in macrophages was, supposedly, the stimulation of sphigmomyelinase activity by a complex of anti-inflammatory cytokines produced by these cells on their activation.     

Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1�CE2F pathway activity

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single‐ and double‐knockout mutations and RNA‐interference (RNAi)‐silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non‐viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma‐related 1 (RBR1)–E2FB complex and this is partly mediated by its N‐terminal LVxCxE motif. Moreover, the S6K1–RBR1 association regulates RBR1 nuclear localization, as well as E2F‐dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient‐limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.     

Molecular Mechanisms of Stabilization of Proteolytic Enzymes: A Model of Thermolysin-Like Microbial Metalloproteases

The molecular mechanisms that ensure the stability of proteolytic proteins are discussed. The autolytic pathway of protease degradation is emphasized. Experiments aimed at increasing the thermal stability of thermolysin-like metalloproteases are comprehensively described.     

Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production

NADPH oxidases are major sources of superoxide (O2*-) and hydrogen peroxide (H2O2) in vascular cells. Production of these reactive oxygen species (ROS) is essential for cell proliferation and differentiation, while ROS overproduction has been implicated in hypertension and atherosclerosis. It is known that the heme-containing catalytic subunits Nox1 and Nox4 are responsible for oxygen reduction in vascular smooth muscle cells from large arteries. However, the exact mechanism of ROS production by NADPH oxidases is not completely understood. We hypothesized that Nox1 and Nox4 play distinct roles in basal and angiotensin II (AngII)-stimulated production of O2*- and H2O2. Nox1 and Nox4 expression in rat aortic smooth muscle cells (RASMCs) was selectively reduced by treatment with siNox4 or antisense Nox1 adenovirus. Production of O2*- and H2O2 in intact RASMCs was analyzed by dihydroethidium and Amplex Red assay. Activity of NADPH oxidases was measured by NADPH-dependent O2*- and H2O2 production using electron spin resonance (ESR) and 1-hydroxy-3-carboxypyrrolidine (CPH) in the membrane fraction in the absence of cytosolic superoxide dismutase. It was found that production of O2*- by quiescent RASMC NADPH oxidases was five times less than H2O2 production. Stimulation of cells with AngII led to a 2-fold increase of O2*- production by NADPH oxidases, with a small 15 to 30% increase in H2O2 formation. Depletion of Nox4 in RASMCs led to diminished basal H2O2 production, but did not affect O2*- or H2O2 production stimulated by AngII. In contrast, depletion of Nox1 in RASMCs inhibited production of O2*- and AngII-stimulated H2O2 in the membrane fraction and intact cells. Our data suggest that Nox4 produces mainly H2O2, while Nox1 generates mostly O2*- that is later converted to H2O2. Therefore, Nox4 is responsible for basal H2O2 production, while O2*- production in nonstimulated and AngII-stimulated cells depends on Nox1. The difference in the products generated by Nox1 and Nox4 may help to explain the distinct roles of these NADPH oxidases in cell signaling. These findings also provide important insight into the origin of H2O2 in vascular cells, and may partially account for the limited pharmacological effect of antioxidant treatments with O2*- scavengers that do not affect H2O2.     

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc.     

Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.     

Noncompetitive immunochemical determination of ribonuclease using transition metal ions and the effect of catalytic hydrogen release

A noncompetitive variant of immunochemical ribonuclease (RNase) determination has been developed, involving the use of Co(II) as a label. A variety of approaches to labeling the immunological reagent with the metal have been assessed. In the variant proposed, catalytic hydrogen release was used as a means of detecting the label, the amount of which was proportional to RNase concentration. Conditions making it possible to record catalytic hydrogen release fluxes were determined. In the presence of RNase, the electrocatalytic effect was maximum at a concentration of Co(II) in the ammoniac buffer, equal to 2 x 10(-4) M (pH 10.0). The dependence was linear in the range 4-2000 ng/ml RNase concentrations (threshold concentration, 2 ng/ml).     

Effects of chloroquine on lysosomes and endocytosis by liver cells in vivo.

1. Chloroquine accumulation in rat liver after a single and repeated drug administration and lysosomal changes resembling some symptoms of lysosomal storage diseases were observed. 2. Repeated chloroquine treatment of rats resulted in increased activity of liver lysosomal enzymes acid phosphatase and beta-galactosidase and a significant enhancement of the activities of cathepsin D and cysteine proteinases were found. 3. No changes in the activity of liver macrophages (as assessed by the colloidal carbon clearance test) or in fluid-phase endocytosis of the marker 125I-polyvinyl-pyrrolidone by hepatocytes in vivo were found.