Intracellular glutathione protects human monocyte-derived macrophages from hypochlorite damage

AimsMacrophages must function in an inflammatory environment of high oxidative stress due to the production of various oxidants. Hypochlorous acid (HOCl) is a potent cytotoxic agent generated by neutrophils and macrophages within inflammatory sites. This study determines whether glutathione is the key factors governing macrophage resistance to HOCl.Main methodsHuman monocyte derived macrophages (HMDM) were differentiated from human monocytes prepared from human blood. The HMDM cells were exposed to micromolar concentrations of HOCl and the timing of the cell viability loss was measured. Cellular oxidative damage was measured by loss of glutathione, cellular ATP, tyrosine oxidation, and inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Key findingsHOCl causes a rapid loss in HMDM cell viability above threshold concentrations. The cell death occurred within 10 min of treatment with the morphological characteristics of necrosis. The HOCl caused the extensive cellular protein oxidation with the loss of tyrosine residue and inactivation of GAPDH, which was accompanied with the loss of cellular ATP. This cellular damage was only observed after the loss of intracellular GSH from the cell. Removal of intracellular GSH with diethyl maleate (DEM) increased the cells' sensitivity to HOCl damage while protecting the intracellular GSH pool with the antioxidant 7,8-dihydroneopterin prevented the HOCl mediated viability loss. Variations in the HOCl LD₅₀ for inducing cell death were strongly correlated with initial intracellular GSH levels.SignificanceIn HMDM cells scavenging of HOCl by intracellular glutathione is sufficient to protect against oxidative loss of key metabolic functions within the cells.     

Adventitious bud induction in Eucalyptus globulus Labill

Summary Adventitious buds and shoots of Eucalyptus globulus Labill. (Tasmanian Bluegum) have been regenerated from cotyledons and hypocotyls from mature embryos and seedlings. Adventitious buds, were induced at high frequency with 0.05 μM thidiazuron in combination with 0.2 μM 2,4-dichlorophenoxyacetic acid or 5 μM α-naphthaleneacetic acid. Culture of explants in the dark inhibited bud induction, but up to 86% of cotyledons, longitudinally split just prior to culture, produced adventitious buds, in the light. Development of buds into shoots occurred only at low frequency, after transfer to media containing N⁶-benzylaminopurine.     

Aversion and attraction to harmful plant secondary compounds jointly shape the foraging ecology of a specialist herbivore

Most herbivorous insect species are restricted to a narrow taxonomic range of host plant species. Herbivore species that feed on mustard plants and their relatives in the Brassicales have evolved highly efficient detoxification mechanisms that actually prevent toxic mustard oils from forming in the bodies of the animals. However, these mechanisms likely were not present during the initial stages of specialization on mustard plants ~100 million years ago. The herbivorous fly Scaptomyza nigrita (Drosophilidae) is a specialist on a single mustard species, bittercress (Cardamine cordifolia; Brassicaceae) and is in a fly lineage that evolved to feed on mustards only in the past 10–20 million years. In contrast to many mustard specialists, S. nigrita does not prevent formation of toxic breakdown products (mustard oils) arising from glucosinolates (GLS), the primary defensive compounds in mustard plants. Therefore, it is an appealing model for dissecting the early stages of host specialization. Because mustard oils actually form in the bodies of S. nigrita, we hypothesized that in lieu of a specialized detoxification mechanism, S. nigrita may mitigate exposure to high GLS levels within plant tissues using behavioral avoidance. Here, we report that jasmonic acid (JA) treatment increased GLS biosynthesis in bittercress, repelled adult female flies, and reduced larval growth. S. nigrita larval damage also induced foliar GLS, especially in apical leaves, which correspondingly displayed the least S. nigrita damage in controlled feeding trials and field surveys. Paradoxically, flies preferred to feed and oviposit on GLS‐producing Arabidopsis thaliana despite larvae performing worse in these plants versus non‐GLS‐producing mutants. GLS may be feeding cues for S. nigrita despite their deterrent and defensive properties, which underscores the diverse relationship a mustard specialist has with its host when lacking a specialized means of mustard oil detoxification.     

p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups

We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-DOPE could serve as a very convenient tool for protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparation of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monoclonal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibody 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whether the specific activity of these immobilized proteins is preserved. The method permits the binding of several dozens protein molecules per single 200 nm liposome. All bound proteins completely preserve their specific activity. Lectin-liposomes are agglutinated by the appropriate polyvalent substrates (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-liposomes specifically bind with biotin-agarose; antibody-liposomes demonstrate high specific binding to the substrate monolayer both in the direct binding assay and in ELISA. A comparison of the suggested method with the method of direct membrane incorporation was made. The effect of the concentration of liposome-grafted PEG on the preservation of specific protein activity in different coupling protocols was also investigated. It was also shown that pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum.     

The redox regulation of intermediary metabolism by a superoxide-aconitase rheostat

In this article, we discuss a hypothesis to explain the preferential synthesis of the superoxide sensitive form of aconitase in mitochondria and the phenotype observed in manganese superoxide dismutase mutant mice, which show a gross over accumulation of stored fat in liver. The model proposes that intermediary metabolism is redox regulated by mitochondrial superoxide generated during mitochondrial respiration. This regulates the level of reducing equivalents (NADH) entering the electron transport chain (ETC) through the reversible inactivation of mitochondrial aconitase. This control mechanism has a dual function; firstly, it regulates levels of superoxide generated by the ETC and, secondly, it fine-tunes metabolism by channeling citrate either for the production of NADH for energy metabolism or diverting it for the synthesis of fats. In this setting, the mitochondrial redox state influences metabolic decisions via a superoxide-aconitase rheostat.