t-Butyl hydroperoxide alters fatty acid incorporation into erythrocyte membrane phospholipid

Because the ability of cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be an important determinant of the ability of cells to tolerate oxidative stress, incorporation of exogenous fatty acid into phospholipid by human erythrocytes has been examined following exposure of the cells to t-butyl hydroperoxide. Exposure of human erythrocytes to t-butyl hydroperoxide (0.5-1.0 mM) results in oxidation of glutathione, formation of malonyldialdehyde, and oxidation of hemoglobin to methemoglobin. Under these conditions, incorporation of exogenous [9,10-3H]oleic acid into phosphatidylethanolamine is enhanced while incorporation of [9,10-3H]oleic acid into phosphatidylcholine is decreased. These effects of t-butyl hydroperoxide on [9,10-3H]oleic acid incorporation are not affected by dissipating transmembrane gradients for calcium and potassium. When malonyldialdehyde production is inhibited by addition of ascorbic acid, t-butyl hydroperoxide still decreases [9,10-3H]oleic acid incorporation into phosphatidylcholine but no stimulation of [9,10-3H]oleic acid incorporation into phosphatidylethanolamine occurs. In cells pre-treated with NaNO2 to convert hemoglobin to methemoglobin, t-butyl hydroperoxide reduces [9,10-3H]oleic acid incorporation into phosphatidylcholine by erythrocytes but does not stimulate [9,10-3H]oleic acid incorporation into phosphatidylethanolamine. Under these conditions oxidation of erythrocyte glutathione and formation of malonyldialdehyde still occur. These results indicate that membrane phospholipid fatty acid turnover is altered under conditions where peroxidation of membrane phospholipid fatty acids occurs and suggest that the oxidation state of hemoglobin influences this response.     

Hyperbaric hyperoxia reversibly inhibits erythrocyte phospholipid fatty acid turnover

The present study is one component of a comprehensive investigation of oxygen tolerance of tissues and organs in normal human subjects. The focus of this study was the acylation of membrane phospholipid in situ by erythrocytes. Activation of exogenous [9,10-3H]oleic acid to acyl thioester and transesterification of the acyl thioester into phospholipid by intact human erythrocytes incubated in vitro decreased 30% after exposure of 10 human subjects to hyperbaric hyperoxia (100% O2, 3 ATA, 3.5 h). Partial recovery of activity could be detected when additional cells were obtained from these subjects and assayed in vitro 24 h after cessation of exposure. No significant change in membrane phospholipid fatty acid composition was detected under these conditions. The reduced glutathione content of intact erythrocytes increased by 15% after hyperbaric hyperoxia and remained elevated 24 h after exposure. In isolated membranes prepared from the same cells activation of [9,10-3H]oleic acid to acyl thioester and its transesterification into phospholipid did not change after hyperoxia. Since the ability of intact cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be necessary for the maintenance of membrane integrity during exposure to oxidative stress, the decrease in [9,10-3H]oleic acid incorporation by human erythrocytes detected in vitro after hyperbaric hyperoxia in vivo may reflect an early event in the pathogenesis of oxygen-induced cellular injury and may be a useful index for assessment of the tolerance of tissues to hyperoxia.     

Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor receptor-mediated Rac activation

Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin alpha1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by alpha1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin alpha1-null mesangial cells produce excessive ROS. Integrin alpha1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in alpha1-null mesangial cells. Thus, our study demonstrates that integrin alpha1beta1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.     

Changes in species composition of European acid grasslands observed along a gradient of nitrogen deposition

Question: Which environmental variables affect floristic species composition of acid grasslands in the Atlantic biogeographic region of Europe along a gradient of atmospheric N deposition? Location: Transect across the Atlantic biogeographic region of Europe including Ireland, Great Britain, Isle of Man, France, Belgium, The Netherlands, Germany, Norway, Denmark and Sweden. Materials and Methods: In 153 acid grasslands we assessed plant and bryophyte species composition, soil chemistry (pH, base cations, metals, nitrate and ammonium concentrations, total C and N, and Olsen plant available phosphorus), climatic variables, N deposition and S deposition. Ordination and variation partitioning were used to determine the relative importance of different drivers on the species composition of the studied grasslands. Results: Climate, soil and deposition variables explained 24% of the total variation in species composition. Variance partitioning showed that soil variables explained the most variation in the data set and that climate and geographic variables accounted for slightly less variation. Deposition variables (N and S deposition) explained 9.8% of the variation in the ordination. Species positively associated with N deposition included Holcus mollis and Leontodon hispidus. Species negatively associated with N deposition included Agrostis curtisii, Leontodon autumnalis, Campanula rotundifolia and Hylocomium splendens. Conclusion: Although secondary to climate gradients and soil biogeochemistry, and not as strong as for species richness, the impact of N and S deposition on species composition can be detected in acid grasslands, influencing community composition both directly and indirectly, presumably through soil‐mediated effects.     

A synthesis of methane emissions from 71 northern,temperate, and subtropical wetlands

Wetlands are the largest natural source of atmospheric methane. Here, we assess controls on methane flux using a database of approximately 19 000 instantaneous measurements from 71 wetland sites located across subtropical, temperate, and northern high latitude regions. Our analyses confirm general controls on wetland methane emissions from soil temperature, water table, and vegetation, but also show that these relationships are modified depending on wetland type (bog, fen, or swamp), region (subarctic to temperate), and disturbance. Fen methane flux was more sensitive to vegetation and less sensitive to temperature than bog or swamp fluxes. The optimal water table for methane flux was consistently below the peat surface in bogs, close to the peat surface in poor fens, and above the peat surface in rich fens. However, the largest flux in bogs occurred when dry 30‐day averaged antecedent conditions were followed by wet conditions, while in fens and swamps, the largest flux occurred when both 30‐day averaged antecedent and current conditions were wet. Drained wetlands exhibited distinct characteristics, e.g. the absence of large flux following wet and warm conditions, suggesting that the same functional relationships between methane flux and environmental conditions cannot be used across pristine and disturbed wetlands. Together, our results suggest that water table and temperature are dominant controls on methane flux in pristine bogs and swamps, while other processes, such as vascular transport in pristine fens, have the potential to partially override the effect of these controls in other wetland types. Because wetland types vary in methane emissions and have distinct controls, these ecosystems need to be considered separately to yield reliable estimates of global wetland methane release.     

Nitrogen deposition and reduction of terrestrial biodiversity: Evidence from temperate grasslands

Biodiversity is thought to be essential for ecosystem stability, function and long-term sustainability. Since nitrogen is the limiting nutrient for plant growth in many terrestrial ecosystems, reactive nitrogen has the potential to reduce the diversity of terrestrial vegetation and associated biota through favouring species adapted to quickly exploiting available nutrients. Although the potential has long been recognised, only recently has enough evidence come together to show beyond reasonable doubt that these changes are already occurring. Linked together, experimental, regional/empirical, and time-series research provide a powerful argument that enhanced deposition of reactive nitrogen across Great Britain, and potentially the rest of Europe, has resulted in a significant and ongoing decline in grassland species richness and diversity.     

Winter fluxes of methane from Minnesota peatlands

Winter fluxes of methane were investigated in northern Minnesota during 1988–89 and 1989–90. Two bogs and a fen emitted methane throughout the snow-covered season (November through March). Fluxes decreased to a low level of 3–16 mg CH₄ m^–2 d^–1 in late March, reflecting decreasing peat temperatures and (in 1989–90) increasing depth of frost in the peat. Winter fluxes calculated by integration for an open poor fen, an open bog, a forested bog hollow, and a hummock site in the forested bog averaged 49, 12, 13, and 5 mg m^–2 d^–1, respectively, in 1989–1990 (the year most measurements were made). These comprised 11%, 4%, 15%, and 21% of total annual flux.     

p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration

Internalization and proteolytic degradation of epidermal growth factor (EGF) receptor (R) following ligand binding is an important mechanism for regulating EGF-stimulated signals. Using pharmacological and RNA interference inhibition of p38 mitogen-activated protein kinase, we show that p38 is required for efficient EGF-induced EGFR destruction but not internalization. In the absence of p38 activity, EGF fails to stimulate the ubiquitin ligase Cbl or ubiquitinylation of EGFR, and internalized EGFR accumulates in intracellular vesicles containing caveolin-1. These effects are accompanied by loss of EGFR phosphorylation on Y1045, a phosphorylation site required for Cbl activation. Furthermore, similar to cells treated with p38 inhibitors, intestinal epithelial cells expressing Y1045F EGFR mutants show increased proliferation but not migration in response to EGF, thus uncoupling these biological responses. Together these data position p38 as a modulator of ligand-stimulated EGFR processing and demonstrate that this processing has a profound impact on the cellular outcome of EGFR signaling.     

The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.     

Enhanced sensitivity to calcium in Duchenne muscular dystrophy.

The ionophore A23187 causes an increase in the Ca content of human erythrocytes and a Ca-dependent increase in K efflux (Gardos effect). These changes are associated with a reduction in osmotic fragility and cell size. Treatment of erythrocytes from patients with Duchenne muscular dystrophy with A23187 results in ⁴⁵Ca uptake comparable to that of erythrocytes from control subjects. However, the reduction in osmotic fragility and K content observed in dystrophic erythrocytes is twofold greater than in control erythrocytes. These results indicate that an alteration in the regulation of erythrocyte membrane function by Ca occurs in Duchenne muscular dystrophy. This alteration may be responsible for other changes in erythrocyte membrane properties observed in Duchenne muscular dystrophy.