232 research outputs found

    Polyethylene terephthalate nanoparticles effect on RAW 264.7 macrophage cells

    Get PDF
    AbstractPlastic pollution is a major environmental concern due to its pervasiveness which continues to increase year on year, as a result of a continuing acceleration in global plastic production and use. Polyethylene terephthalate (PET) is among the most produced plastics, commonly used as food and beverage containers. Once released in the environment, the degradation of plastic materials produces micro-and nano-plastics, with a particular concern about potential toxicological effects if they cross epithelial barriers via inhalation or ingestion. In this work, the effect of PET nanoparticles (PET-NPs) (≤ 250 d.nm) was assayed on mouse macrophages cell line (RAW 264.7) in in vitro experiments. Results showed that PET nanoparticles were easily internalized by the cells, 15 μg/mL of nanoparticles concentration had exhibited effects in cell proliferation and a slightly increased production of Reactive Oxygen Species (ROS), which seems to trigger cell response as foreign particles related to upregulation of PCDH12, IGH-V10, ROBO1 genes, and cell maintenance functions, related to FTSJ2 gene upregulation. Thus, the RAW 264.7 results showed here are useful towards for a preliminary and understanding of the potentially toxic effects related to PET nanoparticles and complementary to other in vitro assays, as the first step into the development of the risk assessment framework

    A Nitrite Biosensor Based on Co-immobilization of Nitrite Reductase and Viologen-modified Chitosan on a Glassy Carbon Electrode

    Get PDF
    An electrochemical nitrite biosensor based on co-immobilization of copper- containing nitrite reductase (Cu-NiR, from Rhodopseudomonas sphaeroides forma sp. denitrificans) and viologen-modified chitosan (CHIT-V) on a glassy carbon electrode (GCE) is presented. Electron transfer (ET) between a conventional GCE and immobilized Cu-NiR was mediated by the co-immobilized CHIT-V. Redox-active viologen was covalently linked to a chitosan backbone, and the thus produced CHIT-V was co-immobilized with Cu-NiR on the GCE surface by drop-coating of hydrophilic polyurethane (HPU). The electrode responded to nitrite with a limit of detection (LOD) of 40 nM (S/N = 3). The sensitivity, linear response range, and response time (t90%) were 14.9 nA/μM, 0.04−11 μM (r2 = 0.999) and 15 s, respectively. The corresponding Lineweaver-Burk plot showed that the apparent Michaelis-Menten constant (KMapp) was 65 μM. Storage stability of the biosensor (retaining 80% of initial activity) was 65 days under ambient air and room temperature storage conditions. Reproducibility of the sensor showed a relative standard deviation (RSD) of 2.8% (n = 5) for detection of 1 μM of nitrite. An interference study showed that anions commonlyfound in water samples such as chlorate, chloride, sulfate and sulfite did not interfere with the nitrite detection. However, nitrate interfered with a relative sensitivity of 64% and this interference effect was due to the intrinsic character of the NiR employed in this study

    Hydrocarbon productivities in different Botryococcus strains: comparative methods in product quantification

    Get PDF
    Six different strains of the green microalgae Botryococcus belonging to the A-race or B-race, accumulating alkadiene or botryococcene hydrocarbons, respectively, were compared for biomass and hydrocarbon productivities. Biomass productivity was assessed gravimetrically upon strain growth in the laboratory under defined conditions. Hydrocarbon productivities were measured by three different and independent experimental approaches, including density equilibrium of the intact cells and micro-colonies, spectrophotometric analysis of hydrocarbon extracts, and gravimetric quantitation of eluted hydrocarbons. All three hydrocarbon-quantitation methods yielded similar results for each of the strains examined. The B-race microalgae Botryococcus braunii var. Showa and Kawaguchi-1 constitutively accumulated botryococcene hydrocarbons equivalent to 30% and 20%, respectively, of their overall biomass. The A-race microalgae Botryococcus braunii, varieties Yamanaka, UTEX 2441 and UTEX LB572 constitutively accumulated alkadiene hydrocarbons ranging from 14% to 13% and 10% of their overall biomass, respectively. Botryococcus sudeticus (UTEX 2629), a morphologically different green microalga, had the lowest hydrocarbon accumulation, equal to about 3% of its overall biomass. Results validate the density equilibrium and spectrophotometric analysis methods in the quantitation of botryococcene-type hydrocarbons. These analytical advances will serve in the screening and selection of B. braunii and of other microalgae in efforts to identify those having a high hydrocarbon content for use in commercial applications

    Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from <i>Bacillus clausii</i>

    Get PDF
    The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization

    Comparison of the structural dynamic and mitochondrial electron-transfer properties of the proapoptotic human cytochrome c variants, G41S, Y48H and A51V

    Get PDF
    Mitochondrial cytochrome c is associated with electron transfer in the respiratory chain and in apoptosis. Four cytochrome c variants have been identified in families that suffer from mild autosomal dominant thrombocytopenia, a platelet disorder associated with increased apoptosis. Three out of the four substitutions, G41S, Y48H and A51V are located on the 40–57 Ω-loop. The G41S and Y48H variants perturb key physicochemical and dynamic properties that result in enhanced functional features associated with apoptotic activity. Herein we characterise the ferric A51V variant. We show by chemical denaturation that this variant causes the native state to be destabilized. Through azide binding kinetics, the population of a pentacoordinate heme form, whereby the Met80 axial ligand is dissociated, is estimated to be of equal magnitude to that found in the Y48H variant. This pentacoordinate form gives rise to peroxidase activity, which despite the similar pentacoordinate population of the A51V variant to that of the Y48H variant, the peroxidase activity of the A51V variant is suppressed. Far-UV circular dichroism spectroscopy and pH jump studies, suggest that a combination of structural and dynamic features in addition to the population of the pentacoordinate form regulate peroxidase activity in these disease variants. Additionally, the steady-state ratio of ferric/ferrous cytochrome c when in turnover with cytochrome c oxidase has been investigated for all 40–57 Ω-loop variants. These studies show that the lower pKa of the alkaline transition for the disease causing variants increases the ferric to ferrous heme ratio, indicating a possible influence on respiration in vivo
    corecore