Fedor Čiampor

Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with β-catenin

Abstract Carbonic anhydrase IX (CA IX) is a cancer-associated transmembrane isoform of zinc metalloenzymes that catalyse interconversion between carbon dioxide and bicarbonate. CA IX is strongly induced by tumor hypoxia and has been proposed to participate in acidification of tumor microenvironment and in cell adhesion. To elucidate the cell adhesion-related role of CA IX, we investigated its subcellular localization and relationship to E-cadherin, a key adhesion molecule whose loss or destabilization is linked to tumor invasion. For this purpose, we generated MDCK cells with constitutive expression of human CA IX protein. During the monolayer formation, CA IX was localized to cell–cell contacts and its distribution in lateral membranes overlapped with E-cadherin. Calcium switch-triggered disruption and reconstitution of cell contacts resulted in relocalization of both CA IX and E-cadherin to cytoplasm and back to plasma membrane. A similar phenomenon was observed in hypoxia-treated and reoxygenated cells. Moreover, CA IX-expressing MDCK cells exhibited reduced cell adhesion capacity and lower levels of Triton-insoluble E-cadherin. Finally, CA IX was found to coprecipitate with β-catenin. We conclude that CA IX has a capacity to modulate E-cadherin-mediated cell adhesion via interaction with β-catenin, which could be of potential significance in hypoxia-induced tumor progression.

Evidence that the amantadine-induced, M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans golgi compartment

Amantadine treatment of cells infected with H7 strains of influenza A viruses causes an M2 protein-mediated conversion of hemagglutinin (HA) from its native to its low pH conformation. Immunofluorescence and electron microscopic observations showed that the structural alteration and hence drug action occur shortly after HA exits from the Golgi complex during its passage through the strans Golgi region. Using the DAMP/anti-DNP pH probe it is evident that virus infection causes increased acidity of the trans Golgi region and that vesicles containing low pH HA in amantadine-treated virus-infected cells are particularly acidic. These results indicate therefore that the alteration in HA is the direct consequence of exposure to an adverse low pH and provide further support for the conclusion that the M2 protein, the target of amantadine action, is involved in regulating vesicular pH, a function important for the correct maturation of the HA glycoprotein.

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles

Abstract Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, 3H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.

Detection and Identification of Coxiella burnetii Based on the Mass Spectrometric Analyses of the Extracted Proteins

Rapid and reliable detection, identification, and typing of bacterial species are necessary in response to natural or terrorist-caused outbreaks of infectious diseases and play crucial roles in diagnosis and efficient treatment. We report here two proteomic approaches with a high potential in the detection and identification of Coxiella burnetii, the causative agent of Q fever. The first of them starts with the acetonitrile (ACN) and trichloroacetic acid extractions of inactivated C. burnetii cells followed by the detection of extracted molecules and ions derived from the inactivated cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the second approach, identification of the proteins extracted by ACN is accomplished after enzymatic digestion by electrospray tandem mass spectrometry coupled to a nanoscale ultraperformance liquid chromatography (LC-MS/MS). In order to observe morphological differences on the surface structures upon extraction, the inactivated and treated cells of the bacterium were examined by electron microscopy. The LC-MS/MS approach has allowed identification of 20 proteins in the ACN extracts of C. burnetii strain RSA 493 that were observed in more than 3 out of 10 experiments.

Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model

Abstract A human blood cell model for immunotoxicity and genotoxicity testing was used to measure the response to polylactic-co-glycolic acid (PLGA-PEO) nanoparticle (NP) (0.12, 3, 15 and 75 μg/cm2 exposure in fresh peripheral whole blood cultures/isolated peripheral blood mononuclear cell cultures from human volunteers (n = 9–13). PLGA-PEO NPs were not toxic up to dose 3 μg/cm2; dose of 75 μg/cm2 displays significant decrease in [3H]-thymidine incorporation into DNA of proliferating cells after 4 h (70% of control) and 48 h (84%) exposure to NPs. In non-cytotoxic concentrations, in vitro assessment of the immunotoxic effects displayed moderate but significant suppression of proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with PWM > CON A, and no changes in PHA cultures. Decrease in proliferative function was the most significant in T-cells stimulated with CD3 antigen (up to 84%). Cytotoxicity of natural killer cells was suppressed moderately (92%) but significantly in middle-dosed cultures (4 h exposure). On the other hand, in low PLGA-PEO NPs dosed cultures, significant stimulation of phagocytic activity of granulocytes (119%) > monocytes (117%) and respiratory burst of phagocytes (122%) was recorded. Genotoxicity assessment revealed no increase in the number of micronucleated binucleated cells and no induction of SBs or oxidised DNA bases in PLGA-PEO-treated cells. To conclude on immuno- and genotoxicity of PLGA-PEO NPs, more experiments with various particle size, charge and composition need to be done.

Proteomic comparison of virulent phase I and avirulent phase II of Coxiella burnetii, the causative agent of Q fever

Coxiella burnetii, a category B biological warfare agent, causes multiple outbreaks of the zoonotic disease Q fever world-wide, each year. The virulent phase I and avirulent phase II variants of the Nine Mile RSA 493 and 439 strains of C. burnetii were propagated in embryonated hen eggs and then purified by centrifugation through Renografin gradients. Total protein fractions were isolated from each phase and subjected to analysis by one-dimensional electrophoresis plus tandem mass spectrometry. A total of 235 and 215 non-redundant proteins were unambiguously identified from the phase I and II cells, respectively. Many of these proteins had not been previously reported in proteomic studies of C. burnetii. The newly identified proteins should provide additional insight into the pathogenesis of Q fever. Several of the identified proteins are involved in the biosynthesis and metabolism of components of the extracellular matrix. Forty-four of the proteins have been annotated as having distinct roles in the pathogenesis or survival of C. burnetii within the harsh phagolysosomal environment. We propose that nine enzymes specifically involved with lipopolysaccharide biosynthesis and metabolism, and that are distinctively present in phase I cells, are virulence-associated proteins.

Afro-Asian cockroach from Chiapas amber and the lost Tertiary American entomofauna

Afro-Asian cockroach from Chiapas amber and the lost Tertiary American entomofauna Cockroach genera with synanthropic species (Blattella, Ectobius, Supella, Periplaneta, Diploptera and ?Blatta), as well as other insects such as honeybees, although natively limited to certain continents nowadays, had circumtropic distribution in the past. The ease of their reintroduction into their former range suggests a post-Early Miocene environmental stress which led to the extinction of cosmopolitan Tertiary entomofauna in the Americas, whilst in Eurasia, Africa and Australia this fauna survived. This phenomenon is demonstrated here on a low diversity (10 spp.) living cockroach genus Supella, which is peculiar for the circumtropical synanthropic brownbanded cockroach S. longipalpa and also for its exclusively free-living cavicolous species restricted to Africa. S. (Nemosupella) miocenica sp. nov. from the Miocene amber of Chiapas in Mexico is a sister species to the living S. mirabilis from the Lower Guinea forests and adjacent savannas. The difference is restricted to the shape of the central macula on the pronotum, and size, which may indicate the around-Miocene origin of the living, extremely polymorphic Supella species and possibly also the isochronic invasion into the Americas. The species also has a number of characteristics of the Asian (and possibly also Australian) uniform genus Allacta (falling within the generic variability of Supella) suggesting Supella is a direct ancestor of the former. The present species is the first significant evidence for incomplete hiati between well defined cockroach genera — a result of the extensive fossil record of the group. The reported specimen is covered by a mycelium of a parasitic fungus Cordyceps or Entomophthora.

Delayed adverse effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether on hypothalamic-pituitary-ovarian axis development and function in Wistar rats.

We studied delayed effects of neonatal exposure to polymeric nanoparticle poly(ethylene glycol)-block-polylactide methyl ether (PEG-b-PLA) on the endpoints related to pubertal development and reproductive function in female Wistar rats from postnatal day 4 (PND4) to PND 176. Female pups were injected intraperitoneally, daily, from PND4 to PND7 with PEG-b-PLA (20 or 40mg/kg b.w.). Both doses of PEG-b-PLA accelerated the onset of vaginal opening compared with the control group. In the low-dose PEG-b-PLA-treated group, a significantly reduced number of regular estrous cycles, increased pituitary weight due to hyperemia, vascular dilatation and congestion, altered course of hypothalamic gonadotropin-releasing hormone-stimulated luteinizing hormone secretion, and increased progesterone serum levels were observed. The obtained data indicate that neonatal exposure to PEG-b-PLA might affect the development and function of hypothalamic-pituitary-ovarian axis (HPO), and thereby alter functions of the reproductive system in adult female rats. Our study indicates a possible neuroendocrine disrupting effect of PEG-b-PLA nanoparticles.

Derived, still living cockroach genus Cariblattoides (Blattida: Blattellidae) from the Eocene sediments of Green River in Colorado, USA

Abstract  Cariblattoides labandeirai sp.n. from the Eocene sediments of Green River in Colorado, USA bear only two plesiomorphies, but also several significant autapomorphies within the advanced and highly derived living cockroach genus. Thus, Cariblattoides with extant occurrence in the Caribbean and South America was historically common in the Nearctic, and represents important evidence for the occurrence of derived living genera of cockroaches ∼50 Ma ago. Generally, the vast majority of living genera were absent during the Palaeocene, thus the diversification of most living cockroach lineages near the Palaeocene/Eocene boundary must have been extremely rapid. Females of living C. suave, the type species, have identical (sophisticated) coloration of pronotum, but the most related living taxa are C. piraiensis and C. fontesi from Brazil (supported by phylogenetical analysis).

Functionalized porous silica&maghemite core-shell nanoparticles for applications in medicine: design, synthesis, and immunotoxicity

Aim To determine cytotoxicity and effect of silica-coated magnetic nanoparticles (MNPs) on immune response, in particular lymphocyte proliferative activity, phagocytic activity, and leukocyte respiratory burst and in vitro production of interleukin-6 (IL-6) and 8 (IL-8), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and granulocyte macrophage colony stimulating factor (GM-CSF). Methods Maghemite was prepared by coprecipitation of iron salts with ammonia, oxidation with NaOCl and modified by tetramethyl orthosilicate and aminosilanes. Particles were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS). Cytotoxicity and lymphocyte proliferative activity were assessed using [3H]-thymidine incorporation into DNA of proliferating human peripheral blood cells. Phagocytic activity and leukocyte respiratory burst were measured by flow cytometry; cytokine levels in cell supernatants were determined by ELISA. Results γ-Fe2O3&SiO2-NH2 MNPs were 13 nm in size. According to TEM, they were localized in the cell cytoplasm and extracellular space. Neither cytotoxic effect nor significant differences in T-lymphocyte and T-dependent B-cell proliferative response were found at particle concentrations 0.12-75 μg/cm2 after 24, 48, and 72 h incubation. Significantly increased production of IL-6 and 8, and GM-CSF cytokines was observed in the cells treated with 3, 15, and 75 µg of particles/cm2 for 48 h and stimulated with pokeweed mitogen (PHA). No significant changes in TNF-α and IFN-γ production were observed. MNPs did not affect phagocytic activity of monocytes and granulocytes when added to cells for 24 and 48 h. Phagocytic respiratory burst was significantly enhanced in the cultures exposed to 75 µg MNPs/cm2 for 48 h. Conclusions The cytotoxicity and in vitro immunotoxicity were found to be minimal in the newly developed porous core-shell γ-Fe2O3&SiO2-NH2 magnetic nanoparticles.