Tomasz Loch

Reduced Mobility of Fibroblast Growth Factor (FGF)-Deficient Myoblasts Might Contribute to Dystrophic Changes in the Musculature of FGF2/FGF6/mdx Triple-Mutant Mice

ABSTRACT Development and regeneration of muscle tissue is a highly organized, multistep process that requires cell proliferation, migration, differentiation, and maturation. Previous data implicate fibroblast growth factors (FGFs) as critical regulators of these processes, although their precise role in vivo is still not clear. We have explored the consequences of the loss of multiple FGFs (FGF2 and FGF6 in particular) for muscle regeneration in mdx mice, which serve as a model for chronic muscle damage. We show that the combined loss of FGF2 and FGF6 leads to severe dystrophic changes in the musculature. We found that FGF6 mutant myoblasts had decreased migration ability in vivo, whereas wild-type myoblasts migrated normally in a FGF6 mutant environment after transplantation of genetically labeled myoblasts from FGF6 mutants in wild-type mice and vice versa. In addition, retrovirus-mediated expression of dominant-negative versions of Ras and Ral led to a reduced migration of transplanted myoblasts in vivo. We propose that FGFs are critical components of the muscle regeneration machinery that enhance skeletal muscle regeneration, probably by stimulation of muscle stem cell migration.

Different extent of cardiac malfunction and resistance to oxidative stress in heterozygous and homozygous manganese-dependent superoxide dismutase-mutant mice

AIMS The mitochondrially expressed manganese-dependent superoxide dismutase (MnSOD, SOD2) is an essential antioxidative enzyme that is necessary for normal heart function. In this study, we investigated the heart function of mice that were exposed to increased oxidative stress for time periods of up to 6 months due to decreased MnSOD activity caused by heterozygous deletion of the MnSOD gene. METHODS AND RESULTS We generated a mouse strain in which the gene encoding MnSOD was exchanged against a cassette containing the SOD cDNA under the control of the tetracycline response element. After breeding with mice carrying the tetracycline receptor, compound mice express MnSOD depending on the presence of tetracycline. Without tetracycline receptor the MnSOD gene is fully inactivated, and animals show an MnSOD-deficient phenotype. Using echocardiographic recordings, we found an impairment of left ventricular functions: MnSOD+/- mice displayed a decrease in fraction shortening and ejection fraction and an increase in left ventricular internal diameter in systole. Furthermore, MnSOD+/- mice developed heart hypertrophy with accompanying fibrosis and necrosis revealed by immunhistochemical analysis. Although we did not find an increase in apoptosis in MnSOD+/- hearts under normal conditions, we observed an increase of the number of apoptotic cells and vascular senescence after treatment with doxorubicin. CONCLUSION Our study demonstrates that lifelong reduction of MnSOD activity has a negative effect on normal heart function. This animal model presents a valuable tool to investigate the mechanism of heart pathology reported in patients bearing different polymorphic variants of the MnSOD gene and to develop new therapeutic strategies through manipulation of the antioxidative defence system.

Repetitive extragenic palindromic PCR (REP-PCR) as a method used for bulking process detection in activated sludge.

Bulking of activated sludge is a world-widely prevalent problem and can lead to loss of bio-oxidation, further deterioration of effluent quality, and even to a complete breakdown of the entire treatment process. Most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria or excess of biopolymers on surface of non-filamentous microbes. Because of complex nature of the bulking phenomenon, the successful bulking control strategy finding is still a very important need awaiting new options and advices. The repetitive extragenic palindromic PCR (REP-PCR) fingerprinting method has been applied to distinguish bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns, using the Ward’s clustering method, have been analyzed to determine homology/similarity relation between particular non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing done based on physicochemical sludge analysis. The choice and application of molecular typing method in sludge analysis will depend upon the needs, skill level, and resources of the laboratory. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be simple, rapid, and effective methods to show differences between population in non-bulking and bulking activated sludge. It is easy to implement, and it may be useful for routinely activated sludge monitoring as well as may be helpful in early detection of bulking process.

Modulation of Porcine Endogenous Retroviruses (PERVs) expression in vitro by shRNA in the presence of cyclosporine A and dexamethasone

BACKGROUND Despite the fact that the risk of Porcine Endogenous Retroviruses (PERV) infection and propagation in human recipients is extremely low, such an event cannot be completely ruled out, especially in immunosuppressed patients. Therefore, the aim of this study was to analyze the expression of PERVs in vitro in the presence of immunosuppression agents: cyclosporine A (CsA), and dexamethasone (DEX). We investigated the possible interactions between immunosuppression drugs, CsA and DEX, and the efficiency of anti-PERV RNAi. MATERIAL/METHODS Plasmid-based vectors expressing shRNAs against all PERV genes were constructed and analyzed. PERVs expression in cultures transfected with anti-PERV RNAi constructions and treated with CsA or DEX was analyzed by Real-Time RT-PCR, Western blot, and by the measurement of RT activity. RESULTS Both CsA and DEX inhibited PERVs expression in cell cultures in vitro. RNAi constructions efficiently knocked down PERV expression in Circe, and de novo PERV-infected HeLa and HEK-293 cell cultures. Pretreatment of Circe cultures with CsA or DEX increased PERVs knockdown by RNAi, but no specific interaction between the drugs and transfection efficiency was observed. CONCLUSIONS Our results demonstrate that cyclosporine A and dexamethasone decrease expression of PERVs in vitro. We also proved that these drugs did not synergize or antagonize RNAi-mediated knockdown of PERVs. These observations may be beneficial in immunosuppressed xenograft recipients; however, due to the controversial literature data concerning influence of immune suppression on graft recipients, our results should be further analyzed.

Contribution of reactive oxygen species to the anticancer activity of aminoalkanol derivatives of xanthone

SummaryReactive oxygen species (ROS) are critically involved in the action of anticancer agents. In this study, we investigated the role of ROS in the anticancer mechanism of new aminoalkanol derivatives of xanthone. Most xanthones used in the study displayed significant pro-oxidant effects similar to those of gambogic acid, one of the most active anticancer xanthones. The pro-oxidant activity of our xanthones was shown both directly (by determination of ROS induction, effects on the levels of intracellular antioxidants, and expression of antioxidant enzymes) and indirectly by demonstrating that the overexpression of manganese superoxide dismutase decreases ROS-mediated cell senescence. We also observed that mitochondrial dysfunction and cellular apoptosis enhancement correlated with xanthone-induced oxidative stress. Finally, we showed that the use of the antioxidant N-acetyl-L-cysteine partly reversed these effects of aminoalkanol xanthones. Our results demonstrated that novel aminoalkanol xanthones mediated their anticancer activity primarily through ROS elevation and enhanced oxidative stress, which led to mitochondrial cell death stimulation; this mechanism was similar to the activity of gambogic acid.

Retraction Note to: Repetitive extragenic palindromic PCR (REP-PCR) as a method used for bulking process detection in activated sludge

Bulking of activated sludge is a world-widely prevalent problem and can lead to loss of bio-oxidation, further deterioration of effluent quality, and even to a complete breakdown of the entire treatment process. Most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria or excess of biopolymers on surface of non-filamentous microbes. Because of complex nature of the bulking phenomenon, the successful bulking control strategy finding is still a very important need awaiting new options and advices. The repetitive extragenic palindromic PCR (REP-PCR) fingerprinting method has been applied to distinguish bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns, using the Ward’s clustering method, have been analyzed to determine homology/similarity relation between particular non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing done based on physicochemical sludge analysis. The choice and application of molecular typing method in sludge analysis will depend upon the needs, skill level, and resources of the laboratory. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be simple, rapid, and effective methods to show differences between population in non-bulking and bulking activated sludge. It is easy to implement, and it may be useful for routinely activated sludge monitoring as well as may be helpful in early detection of bulking process.