Peroxynitrite signaling: receptor tyrosine kinases and activation of stress-responsive pathways

Peroxynitrite, generated for example in inflammatory processes, is capable of nitrating and oxidizing biomolecules, implying a considerable impact on the integrity of cellular structures. Cells respond to stressful conditions by the activation of signaling pathways, including receptor tyrosine kinase-dependent pathways such as mitogen-activated protein kinases and the phosphoinositide-3-kinase/Akt pathway. Peroxynitrite affects signaling pathways by nitration as well as by oxidation: while nitration of tyrosine residues by peroxynitrite modulates signaling processes relying on tyrosine phosphorylation and dephosphorylation, oxidation of phosphotyrosine phosphatases may lead to an alteration in the tyrosine phosphorylation/dephosphorylation balance. The flavanol (-)-epicatechin is a potent inhibitor of tyrosine nitration and may be employed as a tool to distinguish signaling effects due to tyrosine nitration from those that are due to oxidation reactions.     

Oxidant-induced signaling: effects of peroxynitrite and singlet oxygen

Following the requirement for cells to cope with oxidative stress, there are cellular adaptation mechanisms at the level of gene expression. Much of what is known about oxidant-induced signaling in mammalian cells was found in experiments using hydrogen peroxide as an oxidant. However, since the biochemical reactivities of various oxidants significantly differ, 'oxidative stress' is not necessarily identical independent of the oxidant employed to bring it about. Here, the biological actions of peroxynitrite and singlet oxygen are presented, focusing on signaling effects. Peroxynitrite is generated in biological systems in the diffusion-controlled reaction of superoxide with nitrogen monoxide and is thus likely to be produced in the vicinity of activated macrophages. Singlet oxygen is generated by stimulated neutrophils in vivo and may further be generated photochemically, e.g. upon exposure of cells to ultraviolet A radiation. Exposure of cells to either of these oxidants elicits a cellular stress response, entailing the activation of signaling cascades that regulate proliferative and apoptotic responses, such as mitogen-activated protein kinase cascades or the phosphoinositide 3-kinase/Akt cascade. Two mechanisms for the oxidant-induced activation of a signaling cascade may be envisaged: (i) the indirect targeting of the cascade by interrupting negative regulation, and (ii) an activating oxidation of one of the constituting components of the cascade. Examples for both mechanisms in relation to peroxynitrite and singlet oxygen are discussed.     

Sensitive and rapid liquid chromatography-tandem mass spectrometry method for the determination of stavudine in human plasma

A sensitive method for the determination of stavudine in plasma was developed, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. The samples were extracted from plasma with Waters, Sep-Pak Vac, 100 mg, tC(18) solid-phase extraction (SPE) columns. Chromatography was performed on a Supelco Discovery C(18), 5 microm, 150 x 2 mm column with a mobile phase consisting of ammonium acetate (0.01 M)-acetonitrile-methanol (800:100:100, v/v/v) at a flow-rate of 0.3 ml/min. Detection was achieved by an Applied Biosystems API 2000 mass spectrometer (LC-MS-MS) set at unit resolution in the multiple reaction monitoring mode (MRM). Atmospheric pressure chemical ionization (APCI) was used for ion production. The mean recovery for stavudine was 94% with a lower limit of quantification set at 4 ng/ml. This assay method makes use of the increased sensitivity and selectivity of mass spectrometric (MS-MS) detection to allow for a more rapid (extraction and chromatography) and selective method for the determination of stavudine in human plasma than has previously been described.     

A spatial model of tree α-diversity and tree density for the Amazon

Large-scale patterns of Amazonian biodiversity have until now been obscured by a sparse and scattered inventory record. Here we present the first comprehensive spatial model of tree -diversity and tree density in Amazonian rainforests, based on the largest-yet compilation of forest inventories and bolstered by a spatial interpolation technique that allows us to estimate diversity and density in areas that have never been inventoried. These data were then compared to continent-wide patterns of rainfall seasonality. We find that dry season length, while only weakly correlated with average tree -diversity, is a strong predictor of tree density and of maximum tree -diversity. The most diverse forests for any given DSL are concentrated in a narrow latitudinal band just south of the equator, while the least diverse forests for any given DSL are found in the Guayana Shield and Amazonian Bolivia. Denser forests are more diverse than sparser forests, even when we used a measure of diversity that corrects for sample size. We propose that rainfall seasonality regulates tree -diversity and tree density by affecting shade tolerance and subsequently the number of different functional types of trees that can persist in an area.     

The PINE study: rationale and design of a randomised comparison of epidural injection of local anaesthetics and steroids versus care-as-usual to prevent postherpetic neuralgia in the elderly [ISRCTN32866390]

BACKGROUND: Postherpetic neuralgia (PHN) is by far the most common complication of herpes zoster (HZ) and one of the most intractable pain disorders. Since PHN is seen most often in the elderly, the number of patients with this disorder is expected to increase in our ageing society. PHN may last for months to years and has a high impact on the quality of life. The results of PHN treatment are rather disappointing. Epidural injection of local anaesthetics and steroids in the acute phase of HZ is a promising therapy for the prevention of PHN. Since randomised trials on the effectiveness of this intervention are lacking, the PINE (Prevention by epidural Injection of postherpetic Neuralgia in the Elderly) study was set up. The PINE study compares the effectiveness and cost-effectiveness of a single epidural injection of local anaesthetics and steroids during the acute phase of HZ with that of care-as-usual (i.e. antivirals and analgesics) in preventing PHN in elderly patients. METHODS / DESIGN: The PINE study is an open, multicenter clinical trial in which 550 elderly (age >/= 50 yr.) patients who consult their general practitioner in the acute phase of HZ (rash < 7 days) are randomised to one of the treatment groups. The primary clinical endpoint is the presence of HZ-related pain one month after the onset of the rash. Secondary endpoints include duration and severity of pain, re-interventions aiming to treat the existing pain, side effects, quality of life, and cost-effectiveness. CONCLUSION: The PINE study is aimed to quantify the (cost-) effectiveness of a single epidural injection during the acute phase of HZ on the prevention of PHN.     

Selenoprotein P protects low-density lipoprotein against oxidation

Selenoprotein P (SeP) is an extracellular glycoprotein with 8-10 selenocysteines per molecule, containing approximately 50% of total selenium in human serum. An antioxidant function of SeP has been postulated. In the present study, we show that SeP protects low-density lipoproteins (LDL) against oxidation in a cell-free in-vitro system. LDL were isolated from human blood plasma and oxidized with CuCl₂, 2,2'-azobis(2-amidinopropane) (AAPH) or peroxynitrite in the presence or absence of SeP, using the formation of conjugated dienes as parameter for lipid peroxidation. SeP delayed the CuCl₂- and AAPH-induced LDL oxidation significantly and more efficiently than bovine serum albumin used as control. In contrast, SeP was not capable of inhibiting peroxynitrite-induced LDL oxidation. The protection of LDL against CuCl₂- and AAPH-induced oxidation provides evidence for the antioxidant capacity of SeP. Because SeP associates with endothelial membranes, it may act in vivo as a protective factor inhibiting the oxidation of LDL by reactive oxygen species.     

Singlet oxygen-induced attenuation of growth factor signaling: possible role of ceramides

Singlet oxygen, an electronically excited form of molecular oxygen, is a primary mediator of the activation of stress-activated protein kinases elicited by ultraviolet A (UVA; 320-400 nm). Here, the effects of singlet oxygen (₁O₂) on the extracellular signal-regulated kinase (ERK) 1/2 and Akt/protein kinase B pathways were analyzed in human dermal fibroblasts. While basal ERK 1/2 phosphorylation was lowered in cells exposed to either ₁O₂, UVA or photodynamic treatment, Akt was moderately activated by photochemically generated ₁O₂ in a phosphoinositide 3-kinase (PI3K)-dependent fashion, resulting in the phosphorylation of glycogen synthase kinase-3 (GSK3). The activation of ERK 1/2 and Akt as induced by stimulation with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) was inhibited by ₁O₂ generated intracellularly upon photoexcitation of rose Bengal (RB). Photodynamic therapy (PDT)-induced apoptosis is known to be associated with increased formation of ceramides. Likewise, both ₁O₂ and UVA induced ceramide generation in human skin fibroblasts. The attenuation of EGF- and PDGF-induced activation of ERK 1/2 and Akt by ₁O₂ was mimicked by stimulation of fibroblasts with the cell-permeable C₂-ceramide. Interestingly, EGF-induced tyrosine phosphorylation of the EGF receptor was strongly attenuated by ₁O₂ but unimpaired by C₂-ceramide, implying that, although ceramide formation may mediate the above attenuation of ERK and Akt phosphorylation induced by ₁O₂, mechanisms beyond ceramide formation exist that mediate impairment of growth factor signaling by singlet oxygen. In summary, these data point to a novel mechanism of ₁O₂ toxicity: the known ₁O₂-induced activation of proapoptotic kinases such as JNK and p38 is paralleled by the prevention of activation of growth factor receptor-dependent signaling and of anti-apoptotic kinases, thus shifting the balance towards apoptosis.     

Characterization of some strains from human clinical sources which resemble "Leptotrichia sanguinegens": description of Sneathia sanguinegens sp. nov., gen. nov

Three strains of a gram-negative, blood or serum requiring, rod-shaped bacterium recovered from human clinical specimens were characterised by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed the unknown rod-shaped strains are members of the same species as some fastidious isolates recovered from human blood specimens and previously designated "Leptotrichia sanguinegens". Based on phylogenetic and phenotypic evidence, it is proposed that the isolates from human sources be classified in a new genus Sneathia, as Sneathia sanguinegens gen. nov., sp. nov. The type strain of Sneathia sanguinegens is CCUG 41628T.     

Cellular interactions involved in Th cell memory

The cellular interactions involved in maintaining CD4+ T cell memory have hitherto not been identified. In this report, we have investigated the roles played by B cells and dendritic cells (DCs) in this process. We show that long-lasting Th cell memory depends on the presence of B cells, but that direct Ag presentation by B cells is not required. Instead, Ag presentation by DCs is critical for the survival of memory Th cells. DCs presenting specific Ag can be detected in animals long after immunization. These findings support a model in which B cells provide an environment in which Ags may be trapped and retained. This Ag is periodically presented to memory CD4+ T cells by DCs, providing an essential survival signal.