In Vitro Antiproliferative and Antioxidant Effects of Extracts from Rubus caesius

The present study was performed to evaluate the effect of different extracts and subfractions from Rubus caesius leaves on two human colon cancer cell lines obtained from two stages of the disease progression lines HT29 and SW948. Tested samples inhibited the viability of cells, both HT29 and SW948 lines, in a concentration-dependent manner. The most active was the ethyl acetate fraction which, applied at the highest concentration (250 μg/mL), decreased the viability of cells (HT29 and SW948) below 66%. The extracts and subfractions were also investigated for antioxidant activities on DPPH and FRAP assays. All extracts, with the exception of water extract at a dose of 250 μg/mL, almost totally reduced DPPH. The highest Fe3+ ion reduction was shown for the diethyl and ethyl acetate fractions. It was more than 6.5 times higher (at a dose 250 μg/mL) as compared to the control. The LC-MS studies of the analysed preparations showed that all samples contain a wide variety of polyphenolics, among which ellagitannins turned out to be the main constituents with dominant ellagic acid, sanguiin H-6, and flavonol derivatives

Effect of Antibiotic Amphotericin B Combinations with Selected 1,3,4-Thiadiazole Derivatives on RPTECs in an In Vitro Model

4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol (C1) and 4-[5-(naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl] benzene1,3-diol (NTBD) are representative derivatives of the thiadiazole group, with a high antimycotic potential and minimal toxicity against normal human fibroblast cells. The present study has proved its ability to synergize with the antifungal activity of AmB. The aim of this work was to evaluate the cytotoxic effects of C1 or NTBD, alone or in combination with AmB, on human renal proximal tubule epithelial cells (RPTECs) in vitro. Cell viability was assessed with the MTT assay. Flow cytometry and spectrofluorimetric techniques were used to assess the type of cell death and production of reactive oxygen species (ROS), respectively. The ELISA assay was performed to measure the caspase-2, -3, and -9 activity. ATR-FTIR spectroscopy was used to evaluate biomolecular changes in RPTECs induced by the tested formulas. The combinations of C1/NTBD and AmB did not exert a strong inhibitory effect on the viability/growth of kidney cells, as evidenced by the negligible changes in the apoptotic/necrotic rate and caspase activity, compared to the control cells. Both NTBD and C1 displayed stronger anti-oxidant activity when combined with AmB. The relatively low nephrotoxicity of the thiadiazole derivative combinations and the protective activity against AmB-induced oxidative stress may indicate their potential use in the therapy of fungal infections

Regulatory Protein OmpR Influences the Serum Resistance of <i>Yersinia enterocolitica</i> O:9 by Modifying the Structure of the Outer Membrane

<div><p>The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in <i>Yersinia enterocolitica</i> O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the <i>ompR</i> mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of <i>Y. enterocolitica</i>, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (<i>ail</i>, <i>ompX</i>, <i>ompC</i> and <i>flhDC</i>) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the <i>ompR</i> mutant. The serum resistance phenotype of <i>ompR</i> seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the <i>ompR</i> mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of <i>flhDC</i> in <i>trans</i>. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the <i>ompR</i> mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of <i>Y. enterocolitica</i> O:9 to complement-mediated killing through remodeling of the outer membrane.</p></div

The susceptibility of <i>Y. enterocolitica</i> O:9 strains with and without protein Ail to normal human serum.

<p>Strains Ye9 (wt), Ye12 (Ye9 Ail⁻), AR4 (OmpR⁻) and AR7 (AR4 Ail⁻) were grown to exponential phase at 37°C and incubated with 50% NHS at 37°C. Aliquots removed at 0, 15, 30 and 60 min were plated for viability counts (CFU). The numbers of CFU/ml were normalized to the T₀ values as a percentage and then converted to log₁₀ to show the survival rate of the bacteria in NHS. Data are the means ±SD from three independent experiments. The asterisks indicate statistically significant differences between Ye9 and AR4 and their isogenic <i>ail</i> mutant Ye12 (Ail⁻) and AR7 (Ail⁻) (* P<0.001, ** P<0.0001, by one way ANOVA with Tukey's comparison post-test).</p

The susceptibility of <i>Y. enterocolitica</i> O:9 strains lacking YadA protein to normal human serum.

<p>Strains Ye9 (wt) and AR4 (<i>ompR</i>) harboring the virulence plasmid pYV (<i>yadA</i>⁺) and their plasmid-cured derivatives (Ye9c and AR4c, respectively) were grown to exponential phase at 37°C and incubated with 50% NHS at 37°C. Aliquots removed at 0, 15, 30 and 60 min were plated for viability counts (CFU). The numbers of CFU/ml were normalized to the T₀ values as a percentage and then converted to log₁₀ to show the survival rate of the bacteria in NHS. Data are the means ±SD from at least three independent experiments. The Ye9c, pYV⁻ and the AR4c, pYV⁻ asterisks indicate statistically significant difference in comparison with the strains carrying pYV (Ye9 and AR4) (* P<0.001, ** P<0.0001, by one way ANOVA with Tukey's comparison post-test), n.s. not significant.</p

Effect of the OmpR protein on <i>ail</i> promoter activity in <i>Y. enterocolitica</i> O:9 strains.

<p>Strains Ye12 (OmpR⁺) and AR7 (OmpR⁻), representing the respective derivatives of Ye9 and AR4, harboring a transcriptional reporter <i>ail-lacZ</i> fusion, and strain AR7 with plasmid pBR3 expressing OmpR, were grown at 25 or 37°C in LB medium. The activity of β-galactosidase was assayed to evaluate <i>ail</i> expression. Values represent mean β-galactosidase activities, expressed in Miller units ± SD, from three independent experiments. Different letters (a, b) above the columns indicate statistically significant differences (P<0.005, covariance with Tukey correction).</p

The susceptibility of <i>Y. enterocolitica</i> O:9 strains with and without protein OmpC to normal human serum.

<p>Strains Ye9 (wt), OP3 (Ye9 OmpC⁻) and OP3/pBBRC4 (<i>ompC</i> mutant OP3 with plasmid pBBRC4 OmpC⁺) were grown to exponential phase at 37°C and incubated with 50% NHS at 37°C. Aliquots removed at 0, 15, 30 and 60 min were plated for viability counts (CFU). The numbers of CFU/ml were normalized to the T₀ values as a percentage and then converted to log₁₀ to show the survival rate of the bacteria in NHS. Data are the means ±SD from three independent experiments, except for strain OP3 with plasmid pBBRC4 (performed only once). The asterisks indicate statistically significant differences between Ye9 and its <i>ompC</i> mutant OP3 (* P<0.001, ** P<0.0001, by one way ANOVA with Tukey's comparison post-test).</p

Analysis of outer membrane proteins of <i>Y. enterocolitica</i> O:9.

<p><b>A</b>. Outer membrane fractions were isolated from bacterial cells grown in LB broth to an OD₆₀₀ of 1.0 at either 25 or 37°C. Coomassie blue-stained SDS-polyacrylamide gel showing OMPs isolated from strains Ye9 and AR4. The positions of the OmpC/F porins, OmpA and YadA are indicated. MW, molecular weight standards (PageRuler Prestained Protein Ladder). The gel shown is representative of the results of an experiment performed several times. <b>B</b>. Whole-cell lysates of different strains, with or without plasmid pYV (pYV⁺/⁻), grown at 37°C to exponential phase were separated by SDS-PAGE and immunoblot analysis was performed using polyclonal YadA antiserum. YadA protein oligomers and monomers are indicated. This experiment was performed three times with similar results. <b>C</b>. Coomassie blue-stained SDS-polyacrylamide gel of whole-cell lysates of strains used for immunoblot analysis to show loading. MW, molecular weight standards (PageRuler Prestained Protein Ladder).</p

Analysis of LPS of <i>Y. enterocolitica</i> O:9 strains.

<p><b>A</b>. The bacteria were grown in LB medium at 25 or 37°C to an OD₆₀₀ of 1.2. LPS was extracted by the phenol-water method and subjected to electrophoresis in 15% SDS-polyacrylamide gels. The LPS was visualized by silver staining. The gel shown is representative of the results of an experiment performed twice. <b>B</b>. Whole-cell lysates of different strains grown at 25 or 37°C to an OD₆₀₀ of 0.3 were separated by SDS-PAGE and immunoblot analysis was performed using polyclonal antiserum specific for <i>Y. enterocolitica</i> serotype O:9 LPS.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p