Entamoeba histolytica Induces Cell Death of HT29 Colonic Epithelial Cells via NOX1-Derived ROS

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.     

Functional Analysis of TiO2 Nanoparticle Toxicity in Three Plant Species

Titanium dioxide nanoparticles (nano-TiO₂) are manufactured and used worldwide in large quantities. However, phytotoxicity research on nano-TiO₂ has yielded confusing results, ranging from strong toxicity to positive effects. Therefore, in this research, the effects of nano-TiO₂ on the germination and root elongation of seed and seedlings were studied. Additionally, the uptake and physiological responses of mature plants were investigated. Physical chemistry data were analyzed to assess the availability of nano-TiO₂. Finally, a hydroponic system designed to overcome nano-TiO₂ precipitation was used to reproduce the environmental conditions of actual fields. Nano-TiO₂ did not have any effect on seed germination or on most of the plant species tested. Nano-TiO₂ had positive effects on root elongation in some species. No physiological differences in enzyme activities or chlorophyll content were detected, even though the plants absorbed nano-TiO₂. Physical chemistry data showed that nano-TiO₂ agglomerated rapidly and formed particles with much bigger hydrodynamic diameters, even in distilled water and especially in a hydroponic system. Furthermore, agglomerated nano-TiO₂ formed precipitates; this would be more severe in an actual field. Consequently, nano-TiO₂ would not be also readily available to plants and would not cause any significant effects on plants. Our results and other reports suggest that titanium itself is not phytotoxic, even though plants absorb titanium. In conclusion, nano-TiO₂ is not toxic to the three plant species, in vitro or in situ.     

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity

Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.     

Progress in the research of S-adenosyl-l-methionine production

This minireview mainly aims at the study of S-adenosyl-l-methionine (SAM) production by microbial fermentation. A brief introduction of the biological role and application of SAM was presented. In general, SAM production can be improved by breeding of the producing strain through the conventional mutation or genetic engineering approach in the molecular or cellular scale, by optimization of culture conditions in the cellular scale or bioreactor engineering scale, or by multiscale approach. The productivity of SAM fermentation has been improved greatly through the efforts of many researchers using the methods previously mentioned. The SAM-producing strains used extensively are Pichia pastoris and Saccharomyces cerevisiae. The effect of SAM on antibiotic production was also exemplified. The skill and scheme beneficial to the improvement of SAM production involves the enhancement of SAM synthetase (methionine adenosyltransferase) activity and selection of engineered constitutive promoters with appropriate strength; seeking for and eliminating the rate-limiting factors in SAM synthesis, namely, knocking off the genes that transform SAM and l-methionine (L-Met) to cysteine; release the feedback inhibition of SAM to methylenetetrahydrofolate reductase; blocking the transsulfuration pathway by interfering the responsible enzymes; enhancing ATP level through pulsed feeding of glycerol; and optimizing the L-Met feeding strategy. Precise control of gene expression and quantitative assessment of physiological parameters in engineered P. pastoris were highlighted. Finally, a discussion of the prospect of SAM production was presented.     

PEGylated human catalase elicits potent therapeutic effects on H1N1 influenza-induced pneumonia in mice

Therapeutic recombinant human catalase (rhCAT) can quench infection-induced reactive oxygen species (ROS), thereby alleviating the associated tissue damage. Although the intranasal route is efficient to deliver native rhCAT to the lung, the therapeutic effect is limited by rapid elimination from the blood. In this study, we modified rhCAT with the active polymer, polyethylene glycol monomethyl ether (PEG)-5000, and analyzed the pharmacokinetics of PEGylated rhCAT in mice. The high tetra-PEGylation ratio was about 60 %, and PEGylation prolonged the half-life of rhCAT in serum (75 vs. 13.5 min for native rhCAT). The protective effects of PEG-rhCAT were investigated in a mouse model of influenza virus A (H1N1)-associated pneumonia. PEG-rhCAT was more effectively delivered than native rhCAT and was associated with higher survival ratio, less extensive lung injuries, reduced ROS levels, and lower viral replication. Collectively, these findings indicate that PEGylation can enhance the therapeutic efficacy of native rhCAT and suggest that PEGylated rhCAT may represent a novel complement therapy for H1N1 influenza-induced pneumonia.     

A plant‐traits approach to assessing the success of alien weed species in irrigated Mediterranean orchards

In addition to their impact on natural habitats, invasive alien plants can have a significant negative effect on agricultural systems and cause economic losses. Flood‐irrigated orchards in the Mediterranean Basin are vulnerable to the invasion of alien weeds, primarily because of the traditional management practices used in the orchards, which are characterized by high soil moisture during the dry summer period, nutrient availability and high levels of disturbance. This study sought to determine whether their biological traits can explain the success of alien weed species. To answer this question, 408 floristic relevés were conducted in 136 flood‐irrigated orchards on the Plains of Lleida (Catalonia, NE of Spain). Richness and cover of native and alien weeds were compared. Furthermore, a set of biological traits were compared between successful and non‐successful weeds for the whole data and separately between native and alien weeds using logistic regression and classification trees. In flood‐irrigated orchards, alien species covered most of their area, even though the richness of alien species was lower than that of the native species. The most important species were C4 species with seeds dispersed by water, and on the other hand, rosulate and caespitose‐reptant hemicryptophytes with long flowering period. Most of these traits fitted with those of the invasive alien weeds, which were mostly C4 species with seeds dispersed by water. Perennial life form characterized successful native weeds. In this study, we discuss how the traditional management of flood irrigation in fruit‐tree orchards favours invasive alien weeds that have specific traits, acting as a reservoir for the spread of alien weeds into other crops and surrounding riparian habitats. We also propose changing management practices in order to avoid the selection of alien weeds and to promote native species.     

A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria

Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The Michaelis–Menten constant (K_m value) and maximal reaction velocity (V_max values) for exocellulase activity were 12.92 μM and 1.55 × 10⁻⁴ μmol min⁻¹, respectively, and the enzyme was optimally active at pH 5.0 and 37 °C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-β-d-cellobioside (0.3 U mg⁻¹), CMC (105.9 U mg⁻¹), birch wood xylan (132.3 U mg⁻¹), oat spelt xylan (67.9 U mg⁻¹), and 2-hydroxyethyl-cellulose (26.3 U mg⁻¹). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.