Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury

Acute respiratory distress syndrome (ARDS) usually requires symptomatic supportive therapy by intubation and mechanical ventilation with the supplemental use of high oxygen concentrations. Although oxygen therapy represents a life-saving measure, the recent discovery of a critical tissue-protecting mechanism predicts that administration of oxygen to ARDS patients with uncontrolled pulmonary inflammation also may have dangerous side effects. Oxygenation may weaken the local tissue hypoxia-driven and adenosine A2A receptor (A2AR)-mediated anti-inflammatory mechanism and thereby further exacerbate lung injury. Here we report experiments with wild-type and adenosine A2AR-deficient mice that confirm the predicted effects of oxygen. These results also suggest the possibility of iatrogenic exacerbation of acute lung injury upon oxygen administration due to the oxygenation-associated elimination of A2AR-mediated lung tissue-protecting pathway. We show that this potential complication of clinically widely used oxygenation procedures could be completely prevented by intratracheal injection of a selective A2AR agonist to compensate for the oxygenation-related loss of the lung tissue-protecting endogenous adenosine. The identification of a major iatrogenic complication of oxygen therapy in conditions of acute lung inflammation attracts attention to the need for clinical and epidemiological studies of ARDS patients who require oxygen therapy. It is proposed that oxygen therapy in patients with ARDS and other causes of lung inflammation should be combined with anti-inflammatory measures, e.g., with inhalative application of A2AR agonists. The reported observations may also answer the long-standing question as to why the lungs are the most susceptible to inflammatory injury and why lung failure usually precedes multiple organ failure

Identification of Novel Functional Inhibitors of Acid Sphingomyelinase

We describe a hitherto unknown feature for 27 small drug-like molecules, namely functional inhibition of acid sphingomyelinase (ASM). These entities named FIASMAs (Functional Inhibitors of Acid SphingoMyelinAse), therefore, can be potentially used to treat diseases associated with enhanced activity of ASM, such as Alzheimer's disease, major depression, radiation- and chemotherapy-induced apoptosis and endotoxic shock syndrome. Residual activity of ASM measured in the presence of 10 µM drug concentration shows a bimodal distribution; thus the tested drugs can be classified into two groups with lower and higher inhibitory activity. All FIASMAs share distinct physicochemical properties in showing lipophilic and weakly basic properties. Hierarchical clustering of Tanimoto coefficients revealed that FIASMAs occur among drugs of various chemical scaffolds. Moreover, FIASMAs more frequently violate Lipinski's Rule-of-Five than compounds without effect on ASM. Inhibition of ASM appears to be associated with good permeability across the blood-brain barrier. In the present investigation, we developed a novel structure-property-activity relationship by using a random forest-based binary classification learner. Virtual screening revealed that only six out of 768 (0.78%) compounds of natural products functionally inhibit ASM, whereas this inhibitory activity occurs in 135 out of 2028 (6.66%) drugs licensed for medical use in humans

Monoclonal Antibody Epitope Mapping of Plasmodium falciparum Rhoptry Proteins

Plasmodium falciparum rhoptry proteins of the 140/130/110-kDa high molecular weight complex (HMWC) are secreted into the erythrocyte membrane during merozoite invasion. Epitopes of membrane-associated HMWC proteins can be detected using rhoptry-specific antibodies by immunofluorescence assays. Phospholipase treatment of ring-infected intact human erythrocytes, membrane ghosts, and inside-out vesicles results in the release of the HMWC as demonstrated by immunoblotting. We characterized the membrane-associating properties of the 110-kDa protein in more detail. PLA from three different sources; bee venom, Naja naja venom, and porcine pancreas, were examined and all were equally effective in releasing the 1l0-kDa protein. Furthermore, PLA activity was inhibited by o-phenanthroline, quinacrine, maleic anhydride, and partially by p-bromophenacyl bromide, indicating that the activity of PLA is specific. Using sequential protease and phospholipase digestion experiments to map the immunoreactive and functional epitopes of the 110-kDa protein, a 35-kDa protease-resistant protein associated with mouse and human erythrocyte membranes was identified. Limited proteolysis of the 110-kDa protein and analysis by immunoblotting demonstrated several immunoreactive cleavage products, including a highly protease-resistant peptide fragment of approximately 35-kDa which corresponds to the membrane-associated protein. Epitope mapping of the 130-kDa rhoptry protein resulted in a different pattern of cleavage products. Stage-specific metabolic labeling of P. falciparum with [ H] palmitate and [ H] myristate was performed to determine the lipophilic properties of the HMWC. Results showed the incorporation of label into proteins of approximate molecular weight 200 and 45-kDa, predominantly in the late schizont stage. Interestingly, proteins of 140 and 110/100-kDa, corresponding to [ S] methionine-labeled proteins were labeled with [ H]palmitate in ring-infected erythrocyte membranes. However, these proteins were not immunoprecipitated by a rhoptry protein-specific monoclonal antibody, 1B9. Similar label incorporation was not obtained with [ H]myristate. In Triton X-l14 solubility studies, the HMWC proteins partitioned into the aqueous phase, suggesting that they are not integral membrane proteins. In addition, the proteins were extracted by 100 mM Na CO , pH 11.5, and immunoprecipitated by rhoptry-specific antibody. These results suggest that the HMWC proteins may exist in a soluble and membrane bound form. The latter may participate in membrane expansion and the formation of the parasitophorous vacuole during merozoite invasion. © 1993 Academic press, Inc. 2 2 2 2 3 3 3 35 3

Plasmodium falciparum: Effects of Membrane Modulating Agents on Direct Binding of Rhoptry Proteins to Human Erythrocytes

We studied the effects of membrane modulation on the interaction of Plasmodium falciparum rhoptry proteins of 140/130/110 kDa (Rhop-H) with human and mouse erythrocytes. Cells treated with 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylcydopropyl)octanoate, myristoleyl alcohol, and proteins extracted with sublytic concentrations of membrane solubilizing detergents were used in erythrocyte binding assays. Protein binding was evaluated by immunoblotting using Rhop-H- and SERA-specific antisera, 1B9, K15, and 5E3, respectively. Protein binding to liposomes prepared with dipalmitoyl-L-α-phosphatidylcholine (DPPC) or dilauroyl-L-α-phosphatidylcholine (DLPC) was also examined. Our results show that erythrocyte membrane modulation markedly enhanced direct Rhop-H binding to intact human erythrocytes. Binding of SERA to intact human erythrocytes appeared unaffected. Both DPPC and DLPC liposomes had similar Rhop-H and SERA protein binding activities. However, binding to DLPC liposomes was reduced. Rhop-H and SERA extracted with the detergents octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, sodium deoxycholate, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate bound directly to intact human erythrocytes, probably by partitioning hydrophobically into the membranes. Sodium carbonate treatment demonstrated a nonintegral association of Rhop-H with the erythrocyte membrane during invasion. Membrane modulation may expose cryptic phospholipid binding sites in the bilayer. © 1995 Academic press, Inc

A Newly Identified Role for Superoxide in Inflammatory Pain JPET #64154 2 Running Title: Superoxide and hyperalgesia

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