The Role of Bcl-2 and Beclin-1 Complex in "Switching" between Apoptosis and Autophagy in Human Glioma Cells upon LY294002 and Sorafenib Treatment.

BACKGROUND: Gliomas are the most malignant tumors of the central nervous system. One of the factors in their high drug resistance is avoiding programmed death (PCD) induction. This is related to the overexpression of intracellular survival pathways: PI3K-Akt/PKB-mTOR and Ras-Raf-MEK-ERK. Apoptosis and autophagy are co-existing processes due to the interactions between Bcl-2 and beclin-1 proteins. Their complex may be a molecular "toggle-switch" between PCD types. The aim of this research was to investigate the role of Bcl-2:beclin-1 complex in glioma cell elimination through the combined action of LY294002 and sorafenib. METHODS: Drug cytotoxicity was estimated with an MTT test. The type of cell death was evaluated using variant microscopy techniques (fluorochrome staining, immunocytochemistry, and transmission electron microscopy), as well as the Bcl-2:beclin-1 complex formation and protein localization. Molecular analysis of PCD indicators was conducted through immunoblotting, immunoprecipitation, and ELISA testing. SiRNA was used to block Bcl-2 and beclin-1 expression. RESULTS: The results showed the inhibitors used in simultaneous application resulted in Bcl-2:beclin-1 complex formation and apoptosis becoming dominant. This was accompanied by changes in the location of the tested proteins. CONCLUSIONS: "Switching" between apoptosis and autophagy using PI3K and Raf inhibitors with Bcl-2:beclin-1 complex formation opens new therapeutic perspectives against gliomas.

Autophagy of Candida albicans cells after the action of earthworm Venetin-1 nanoparticle with protease inhibitor activity.

The present studies show the effect of the Venetin-1 protein-polysaccharide complex obtained from the coelomic fluid of the earthworm Dendrobaena veneta on Candida albicans cells. They are a continuation of research on the mechanisms of action, cellular targets, and modes of cell death. After the action of Venetin-1, a reduced survival rate of the yeast cells was noted. The cells were observed to be enlarged compared to the controls and deformed. In addition, an increase in the number of cells with clearly enlarged vacuoles was noted. The detected autophagy process was confirmed using differential interference contrast, fluorescence microscopy, and transmission electron microscopy. Autophagic vesicles were best visible after incubation of fungus cells with the Venetin-1 complex at a concentration of 50 and 100 µg mL-1. The changes in the vacuoles were accompanied by changes in the size of mitochondria, which is probably related to the previously documented oxidative stress. The aggregation properties of Venetin-1 were characterized. Based on the results of the zeta potential at the Venetin-1/KCl interface, the pHiep = 4 point was determined, i.e. the zeta potential becomes positive above pH = 4 and is negative below this value, which may affect the electrostatic interactions with other particles surrounding Venetin-1.

Polycatecholamine and gentamicin as modifiers for antibacterial and blood-biocompatible polyester vascular prostheses.

Polyester (PET) prostheses are commonly used in reconstructive vascular surgery. The most serious complication after implantation is early or late infection of the graft. Therefore, there is high demand to protect prosthesis against bacterial adhesion and biofilm development. For this reason, in this work PET prostheses were first coated by highly adhesive polycatecholamine layer. The grafts were then coupled with gentamicin and studied in relation to morphological and structural properties, biological safety (contact with blood, reaction of vascular endothelial cells (HUVEC), Danio rerio fish), drug release and antibacterial activity. Among two tested catecholamine monomers, L-DOPA was found to be more effective precursor in this process than dopamine. For L-DOPA, assistance of Cu2+, Mg2+ and Na+ ions seems to increase the amount of further immobilized drug. Coated prostheses exhibited greater human endothelial cell proliferation increase and lower cytotoxic effect than uncoated. The modification reduced the hemolysis observed for pristine commercial graft and limited the rate of abnormalities in D. rerio larvae, confirming the safety of the proposed modification. The coating allowed to double the amount of immobilized antibiotic in comparison with uncoated graft which resulted in increased antibacterial activity and reduced bacterial adhesion against 4 bacterial strains prevalent in biomaterials infections. Overall, poly(L-DOPA)-coatings deposited on PET vascular grafts can effectively functionalize these prostheses for higher safety in biomedical applications.

Involvement of PI3K Pathway in Glioma Cell Resistance to Temozolomide Treatment.

The aim of the study was to investigate the anticancer potential of LY294002 (PI3K inhibitor) and temozolomide using glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells. Apoptosis, autophagy, necrosis, and granules in the cytoplasm were identified microscopically (fluorescence and electron microscopes). The mitochondrial membrane potential was studied by flow cytometry. The activity of caspases 3, 8, and 9 and Akt was evaluated fluorometrically, while the expression of Beclin 1, PI3K, Akt, mTOR, caspase 12, and Hsp27 was determined by immunoblotting. SiRNA was used to block Hsp27 and PI3K expression. Cell migration and localization of Hsp27 were tested with the wound healing assay and immunocytochemistry, respectively. LY294002 effectively diminished the migratory potential and increased programmed death of T98G and MOGGCCM. Autophagy was dominant in MOGGCCM, while apoptosis was dominant in T98G. LY294002 with temozolomide did not potentiate cell death but redirected autophagy toward apoptosis, which was correlated with ER stress. A similar effect was observed after blocking PI3K expression with siRNA. Transfection with Hsp27 siRNA significantly increased apoptosis related to ER stress. Our results indicate that inhibition of the PI3K/Akt/mTOR pathway sensitizes glioma cells to apoptosis upon temozolomide treatment, which was correlated with ER stress. Hsp27 increases the resistance of glioma cells to cell death upon temozolomide treatment.

Four Types of TiO2 Reduced the Growth of Selected Lactic Acid Bacteria Strains.

Food-grade titanium dioxide (TiO2) containing a nanoparticle fraction (TiO2 NPs -nanoparticles) is widely used as a food additive (E171 in the EU). In recent years, it has increasingly been raising controversies as to the presence or absence of its harmful effects on the gastrointestinal microbiota. The complexity and variability of microbiota species present in the human gastrointestinal tract impede the assessment of the impact of food additives on this ecosystem. As unicellular organisms, bacteria are a very convenient research model for investigation of the toxicity of nanoparticles. We examined the effect of TiO2 (three types of food-grade E171 and one TiO2 NPs, 21 nm) on the growth of 17 strains of lactic acid bacteria colonizing the human digestive tract. Each bacterial strain was treated with TiO2 at four concentrations (60, 150, 300, and 600 mg/L TiO2). The differences in the growth of the individual strains were caused by the type and concentration of TiO2. It was shown that the growth of a majority of the analyzed strains was decreased by the application of E171 and TiO2 NPs already at the concentration of 150 and 300 mg/L. At the highest dose (600 mg/L) of the nanoparticles, the reactions of the bacteria to the different TiO2 types used in the experiment varied.

Complete genome sequence of Lactobacillus rhamnosus Pen, a probiotic component of a medicine used in prevention of antibiotic-associated diarrhoea in children.

BACKGROUND: Lactobacillus rhamnosus Pen is a human endogenous strain with well-documented health promoting properties that is used for production of probiotics. It has a long safety history of application, and its effectiveness in the prevention of antibiotic-associated diarrhoea has also been confirmed in clinical trials. RESULTS: Here we present the complete genome sequence of L. rhamnosus Pen, which consists of a circular 2,884,4966-bp chromosome with a GC content of 46.8%. Within 2907 open reading frames (ORFs), genes involved with probiotic properties were identified. A CRISPR locus, consisting of a 1092-nt region with 16 spacers, was also detected. Finally, an intact prophage of ~ 40.7 kb, 57 ORFs, GC content 44.8% was identified. CONCLUSIONS: Genomic analysis confirmed the probiotic properties of L. rhamnosus Pen and may indicate new biotechnological applications of this industrially important strain.

Rhizobium strains differ considerably in outer membrane permeability and polymyxin B resistance.

Six rhizobium (Rhizobium leguminosarum bv. Trifolii TA1, Sinorhizobium meliloti 1021, Mesorhizobium huakuii IFO 15243(T), Ochrobactrum lupini LUP 21(T), Bradyrhizobium japonicum USDA110 and B. elkanii USDA 76) and two Escherichia coli strains (E. coli ATCC 25922 and E. coli HB 101) were compared in respect to polymyxin B and EDTA resistance, as well as bacterial outer membrane (OM) permeability to a fluorescent hydrophobic agent (N-phenyl-1-naphthylamine - NPN). TEM (Transmission Electron Microscopy) and a microbial test demonstrated that all the rhizobia were much more resistant to polymyxin B in comparison with E. coli strains. EDTA and polymyxin B enhance permeability of B. japonicum and O. lupini OM. Other rhizobia incorporated NPN independently of the presence of membrane-deteriorating agents; however, the level of fluorescence (measured as NPN absorption) was strain dependent.

Spontaneous release of bacteriophage particles by Lactobacillus rhamnosus pen.

The identification of bacteriophage proteins on the surface of Lactobacillus rhamnosus Pen was performed by LC-MS/MS analysis. Among the identified proteins, we found a phage-derived major tail protein, two major head proteins, a portal protein, and a host specificity protein. Electron microscopy of a cell surface extract revealed the presence of phage particles in the analyzed samples. The partial sequence of genes encoding the major tail protein for all tested L. rhamnosus strains was determined with specific primers designed in this study. Next, RT-PCR analysis allowed detection of the expression of the major tail protein gene in L. rhamnosus strain Pen at all stages of bacterial growth. The transcription of genes encoding the major tail protein was also proved for other L. rhamnosus strains used in this study. The present work demonstrates the spontanous release of prophage-encoded particles by a commercial probiotic L. rhamnosus strain, which did not significantly affect the bacterial growth of the analyzed strain.

Gut bacterium of Dendrobaena veneta (Annelida: Oligochaeta) possesses antimycobacterial activity.

The new bacterial strain with antimycobacterial activity has been isolated from the midgut of Dendrobaena veneta (Annelida). Biochemical and molecular characterization of isolates from 18 individuals identified all as Raoultella ornithinolytica genus with 99% similarity. The bacterium is a possible symbiont of the earthworm D. veneta. The isolated microorganism has shown the activity against four strains of fast-growing mycobacteria: Mycobacterium butiricum, Mycobacterium jucho, Mycobacterium smegmatis and Mycobacterium phlei. The multiplication of the gut bacterium on plates with Sauton medium containing mycobacteria has caused a lytic effect. After the incubation of the cell free extract prepared from the gut bacterium with four strains of mycobacteria in liquid Sauton medium, the cells of all tested strains were deformed and divided to small oval forms and sometimes created long filaments. The effect was observed by the use of light, transmission and scanning microscopy. Viability of all examined species of mycobacteria was significantly decreased. The antimycobacterial effect was probably the result of the antibiotic action produced by the gut bacterium of the earthworm. The application of ultrafiltration procedure allowed to demonstrate that antimicrobial substance with strong antimycobacterial activity from bacterial culture supernatant, is a protein with the molecular mass above 100 kDa.

Morphology and general characteristics of bacteriophages infectious to Robinia pseudoacacia mesorhizobia.

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley's classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10-25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100-120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 µg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from approximately 39 kb for group C phages to approximately 80 kb for B2.

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group.

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.

Quercetin suppresses heat shock-induced nuclear translocation of Hsp72.

The effect of quercetin and heat shock on the Hsp72 level and distribution in HeLa cells was studied by Western blotting, indirect immunofluorescence and immunogold electron microscopy. In control cells and after quercetin treatment, Hsp72 was located both in the cytoplasm and in the nucleus in comparable amounts. After hyperthermia, the level of nuclear Hsp72 raised dramatically. Expression of Hsp72 in cytoplasm was also higher but not to such extent as that observed in the nucleus. Preincubation of heated cells with quercetin inhibited strong Hsp72 expression observed after hyperthermia and changed the intracellular Hsp72 distribution. The cytoplasmic level of protein exceeded the nuclear one, especially around the nucleus, where the coat of Hsp72 was noticed. Observations indicating that quercetin was present around and in the nuclear envelope suggested an involvement of this drug in the inhibition of nuclear translocation. Our results indicate that pro-apoptotic activity of quercetin may be correlated not only with the inhibition of Hsp72 expression but also with suppression of its migration to the nucleus.