Mass spectrometry strategies to unveil modified aminophospholipids of biological interest

The biological functions of modified aminophospholipids (APL) have become a topic of interest during the last two decades, and distinct roles have been found for these biomolecules in both physiological and pathological contexts. Modifications of APL include oxidation, glycation, and adduction to electrophilic aldehydes, altogether contributing to a high structural variability of modified APL. An outstanding technique used in this challenging field is mass spectrometry (MS). MS has been widely used to unveil modified APL of biological interest, mainly when associated with soft ionization methods (electrospray and matrix-assisted laser desorption ionization) and coupled with separation techniques as liquid chromatography. This review summarizes the biological roles and the chemical mechanisms underlying APL modifications, and comprehensively reviews the current MS-based knowledge that has been gathered until now for their analysis. The interpretation of the MS data obtained by in vitro-identification studies is explained in detail. The perspective of an analytical detection of modified APL in clinical samples is explored, highlighting the fundamental role of MS in unveiling APL modifications and their relevance in pathophysiology.publishe

Development of a lung slice preparation for recording ion channel activity in alveolar epithelial type I cells

BACKGROUND: Lung fluid balance in the healthy lung is dependent upon finely regulated vectorial transport of ions across the alveolar epithelium. Classically, the cellular locus of the major ion transport processes has been widely accepted to be the alveolar type II cell. Although evidence is now emerging to suggest that the alveolar type I cell might significantly contribute to the overall ion and fluid homeostasis of the lung, direct assessment of functional ion channels in type I cells has remained elusive. METHODS: Here we describe a development of a lung slice preparation that has allowed positive identification of alveolar type I cells within an intact and viable alveolar epithelium using living cell immunohistochemistry. RESULTS: This technique has allowed, for the first time, single ion channels of identified alveolar type I cells to be recorded using the cell-attached configuration of the patch-clamp technique. CONCLUSION: This exciting new development should facilitate the ascription of function to alveolar type I cells and allow us to integrate this cell type into the general model of alveolar ion and fluid balance in health and disease

Oxr1 Is Essential for Protection against Oxidative Stress-Induced Neurodegeneration

Oxidative stress is a common etiological feature of neurological disorders, although the pathways that govern defence against reactive oxygen species (ROS) in neurodegeneration remain unclear. We have identified the role of oxidation resistance 1 (Oxr1) as a vital protein that controls the sensitivity of neuronal cells to oxidative stress; mice lacking Oxr1 display cerebellar neurodegeneration, and neurons are less susceptible to exogenous stress when the gene is over-expressed. A conserved short isoform of Oxr1 is also sufficient to confer this neuroprotective property both in vitro and in vivo. In addition, biochemical assays indicate that Oxr1 itself is susceptible to cysteine-mediated oxidation. Finally we show up-regulation of Oxr1 in both human and pre-symptomatic mouse models of amyotrophic lateral sclerosis, indicating that Oxr1 is potentially a novel neuroprotective factor in neurodegenerative disease

Persistent Exposure to Mycoplasma Induces Malignant Transformation of Human Prostate Cells

Recent epidemiologic, genetic, and molecular studies suggest infection and inflammation initiate certain cancers, including those of the prostate. The American Cancer Society, estimates that approximately 20% of all worldwide cancers are caused by infection. Mycoplasma, a genus of bacteria that lack a cell wall, are among the few prokaryotes that can grow in close relationship with mammalian cells, often without any apparent pathology, for extended periods of time. In this study, the capacity of Mycoplasma genitalium, a prevalent sexually transmitted infection, and Mycoplasma hyorhinis, a mycoplasma found at unusually high frequency among patients with AIDS, to induce a malignant phenotype in benign human prostate cells (BPH-1) was evaluated using a series of in vitro and in vivo assays. After 19 weeks of culture, infected BPH-1 cells achieved anchorage-independent growth and increased migration and invasion. Malignant transformation of infected BPH-1 cells was confirmed by the formation of xenograft tumors in athymic mice. Associated with these changes was an increase in karyotypic entropy, evident by the accumulation of chromosomal aberrations and polysomy. This is the first report describing the capacity of M. genitalium or M. hyorhinis infection to lead to the malignant transformation of benign human epithelial cells and may serve as a model to further study the relationship between prostatitis and prostatic carcinogenesis

Proteomic and Physiological Responses of Kineococcus radiotolerans to Copper

Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0–1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R2 = 0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration