Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli

Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H₂O₂ stimulation.     

蛋白激酶组在玉米叶片ABA和H202诱导抗氧化防护中的作用

蛋白磷酸化在植物细胞脱落酸（ABA）介导的信号转导中起重要作用。然而,很多参与ABA信号途径的蛋白元件仍不清楚。使用改进的体外激酶试验方法的研究结果表明,在玉米叶片中,ABA和H2O2能够快速活化蛋白激酶总活性和ca^2＋依赖型蛋白激酶总活性;ABA诱导的蛋白激酶总活性增加可以被活性氧的抑制剂和清除剂抑制,蛋白激酶抑制剂不仅可以降低ABA和H2O2诱导的激酶活性增加,而且也可以弱化它们对抗氧化防护酶活性的诱导作用;ABA和H2O2引发的蛋白磷酸化作用显著居先于它们诱导的抗氧化防护作用。使用凝胶激酶试验方法进行研究发现,一组分子量分别为66kDa,52kDa,49kDa和35kDa的蛋白激酶可能介导了ABA和H2O2诱导的抗氧化防护反应,并且66kDa和49kDa的蛋白激酶可能在ROS的下游起作用,而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

New and highly efficient methodology for screening high-yield strains of cytotoxic deacetylmycoepoxydiene (DAM)

Aims: To establish a highly efficient methodology for screening high yield strains of cytotoxic deacetylmycoepoxydiene (DAM), to meet the need of research on its mechanism of anti‐tumor properties and in vivo toxicity studies. Methods and Results: A simple, sensitive, and highly repetitive screening procedure ‘Antimicrobial‐TLC–HPLC’ (ATH) was established for the rapid obtaining of high‐yielding DAM mutants to replace the time and labor intensive anti‐tumor activity assay (MTT). With this ATH method, four highly yielding DAM mutants were selected out of 5000 total mutants, one of which, M4‐143, showed yields of more than 300 times (250·3 mg l^?1) that of the parent strain A123. Conclusions: The ATH method developed in this work has proven to be both economical and highly efficient with the screening of 1200 mutants in a one week time period, thusly shortening the expenditure of time and labor, without missing a single high‐yield mutant. Due to these characteristics, it is superior to other HTS screening methods described in earlier literature. The mutant M4‐143 has a good genetic stability and can be used for further research. Significance and Impact of the Study: This ATH screening method is not only perfect for screening high‐yield DAM mutants, but also, it is suitable to screen the strain libraries for those strains that have the ability to produce natural metabolites with antitumor activity.     

Measure of stress response induced by temperature and salinity changes on hatched larvae of three marine gastropod species

To better understand the cascade of molecular reactions leading to delayed development and mortality of early life stages of marine intertidal gastropods, in response to temperature and salinity changes associated with climate change, three biomarkers: total antioxidant capacity, lipid peroxidation and lysosomal stability were investigated on hatched larvae. Encapsulated embryos of three marine gastropod species (Bembicium nanum, Siphonaria denticulata and Dolabrifera brazieri), which have already proven responsive to thermal and osmotic variations, were exposed to six combinations of temperature (22 °C and 30 °C) and salinity (25‰, 35‰ and 45‰) until the larvae hatched. Time to hatching was affected by salinity and temperature in all three species. High salinity (45‰) generally retarded the hatching process although the response was species-specific for temperature. Total antioxidant capacity and lipid peroxidation were also highly species-specific with the general trend showing that these biomarkers were adversely affected by high temperature (30 °C) at salinities of 25‰ and 45‰. Bembicium nanum lysosomal destabilisation increased significantly with an increase in temperature and salinity (30 °C and 45‰) and this was associated with delayed development and increased mortality. Investigations on the additional biomarker, lysosomal stability, gave a clearer picture of the numerous and complex molecular and cellular mechanisms leading to mortality and underdevelopment in response to environmental stress for this species. As few differences were observed in the enzymatic biomarkers total antioxidant capacity and lipid peroxidation between hatched larvae and the previously investigated encapsulated embryo response to thermal and osmotic stress, it is suggested that further studies could be undertaken using embryos encapsulated in egg masses, as it is less time consuming than working on hatched larvae.     

THE BIOTRANSFORMATION,BIODEGRADATION, AND BIOREMEDIATION OF ORGANIC COMPOUNDS BY MICROALGAE1

Rapid growth in the biotechnological industry and production has put tremendous pressure on the biological methods that may be used according to the guidelines of green chemistry. However, despite continuing dramatic increases in published research on organic biotransformation by microorganisms, more research exists with microalgae. Our efforts in transforming chemicals such as organic compounds for the production of functionalized products help to lessen the environmental effects of organic synthesis. These biotransformations convert organic contaminants to obtain carbon or energy for growth or as cosubstrates. This review aims to focus on the potential of microalgae in transformation, conversion, remediation, accumulation, degradation, and synthesis of various organic compounds. However, these technologies have the ability to provide the most efficient and environmentally safe approach for inexpensive biotransforming of a variety of organic contaminants, which are most industrial residues. In addition, the recent advances in microalgal bioactivity were discussed.     

Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera)

Nosema ceranae, a microsporidian parasite originally described in the Asian honey bee Apis cerana, has recently been found to be cross-infective and to also parasitize the European honey bee Apis mellifera. Since this discovery, many studies have attempted to characterize the impact of this parasite in A. mellifera honey bees. Nosema species can infect all colony members, workers, drones and queens, but the pathological effects of this microsporidium has been mainly investigated in workers, despite the prime importance of the queen, who monopolizes the reproduction and regulates the cohesion of the society via pheromones. We therefore analyzed the impact of N. ceranae on queen physiology. We found that infection by N. ceranae did not affect the fat body content (an indicator of energy stores) but did alter the vitellogenin titer (an indicator of fertility and longevity), the total antioxidant capacity and the queen mandibular pheromones, which surprisingly were all significantly increased in Nosema-infected queens. Thus, such physiological changes may impact queen health, leading to changes in pheromone production, that could explain Nosema-induced supersedure (queen replacement).     

Antimicrobial peptides increase tolerance to oxidant stress in Drosophila melanogaster

It is well appreciated that reactive oxygen species (ROS) are deleterious to mammals, including humans, especially when generated in abnormally large quantities from cellular metabolism. Whereas the mechanisms leading to the production of ROS are rather well delineated, the mechanisms underlying tissue susceptibility or tolerance to oxidant stress remain elusive. Through an experimental selection over many generations, we have previously generated Drosophila melanogaster flies that tolerate tremendous oxidant stress and have shown that the family of antimicrobial peptides (AMPs) is over-represented in these tolerant flies. Furthermore, we have also demonstrated that overexpression of even one AMP at a time (e.g. Diptericin) allows wild-type flies to survive much better in hyperoxia. In this study, we used a number of experimental approaches to investigate the potential mechanisms underlying hyperoxia tolerance in flies with AMP overexpression. We demonstrate that flies with Diptericin overexpression resist oxidative stress by increasing antioxidant enzyme activities and preventing an increase in ROS levels after hyperoxia. Depleting the GSH pool using buthionine sulfoximine limits fly survival, thus confirming that enhanced survival observed in these flies is related to improved redox homeostasis. We conclude that 1) AMPs play an important role in tolerance to oxidant stress, 2) overexpression of Diptericin changes the cellular redox balance between oxidant and antioxidant, and 3) this change in redox balance plays an important role in survival in hyperoxia.     

Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity

Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide (^•NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO⁻ + ONOOH) formation, a strong oxidant arising from the reaction of ^•NO with superoxide radical (O₂^˙̄). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O₂^˙̄ during a 60–90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained ^•NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when ^•NO and O₂^˙̄ were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite.     

Derivatives of rhodamine 19 as mild mitochondria-targeted cationic uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.     

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.