Hans P. Deigner

Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits.

The objective of the study was to analyze the intracellular antioxidative response of macrophages (MΦ) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit MΦ, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human MΦ with oxidized‐LDL (ox‐LDL) resulted in an induction of MnSOD mRNA production as shown by RT‐PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of MΦ. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=–0.67, P<0.001) and GSH concentration (r=–0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD‐IR in Mac‐1 (CD 11b/CD 18)‐immunoreactive (ir) MΦ of human arteries and, similarly, in RAM‐11‐ir MΦ of rabbit ones. The relation of MnSOD‐ir MΦ decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD‐ir MΦ in atherosclerotic plaques revealed TUNEL‐positive nuclei, indicating DNA strand breaks, and p53‐IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in MΦ in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox‐LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species‐mediated mitochondrial‐dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.—Kinscherf, R., Deigner, H.‐P., Usinger, C., Pill, J., Wagner, M., Kamencic, H., Hou, D., Chen, M., Schmiedt, W., Schrader, M., Kovacs, G., Kato, K., Metz, J. Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits. FASEB J. 11, 1317–1328 (1997)

Anti-platelet drugs and outcome in severe infection: Clinical impact and underlying mechanisms

Platelet activation contributes to microvascular thrombosis and organ failure in systemic inflammation. We tested the hypothesis whether anti-platelet drugs might favourably affect outcome in patients at risk for organ failure as well as in a mouse model of endotoxin shock. Two hundred twenty-four consecutive patients who were admitted for community acquired pneumonia over a time period of 5 years to a University Hospital were enrolled; about 20% of whom received anti-platelet drugs (acetylsalicylic acid, thienopyridines) for secondary prevention of cardiovascular disease. Patients with anti-platelet drugs were about 12 years old but did not differ in SOFA score and routine laboratory parameters at admission. Logistic regression and 2 × 2 table analysis in age-matched subgroups indicated that anti-platelet drugs may reduce the need of intensive care treatment (odds ratio (OR) 0.32 [95% confidential interval: 0.10–1.00] and 0.19 [0.04–0.87], respectively). In age-matched subgroups, the use of anti-platelet drugs was also associated with a shorter stay in hospital (13.9 ± 6.2 vs. 18.2 ± 10.2 days; p < 0.02). In the animal model Balb/c mice were pre-treated with clopidogrel (added to drinking water) for 4 days prior to intraperitoneal (i.p.) administration of endotoxin (lipopolsaccharide (LPS) from Escherichia coli 0111:B4). Within the first 48 hours after LPS there were no differences between clopidogrel and control animals (n = 26 each) in macro-haemodynamics. However, clopidogrel abolished the LPS-induced drop in platelet count and reduced fibrin deposition in lung tissue. Using DNA microarray technology, we could show that clopidogrel suppressed endotoxin-induced up-regulation of inflammation-relevant genes, including arachidonate-5-lipoxygenase activating protein and leukotriene B4 receptor 1. According to our data a possible benefit of anti-platelet drugs in patients on risk for systemic inflammation and organ failure should be tested in a prospective trial.

Modified low density lipoprotein delivers substrate for ceramide formation and stimulates the sphingomyelin‐ceramide pathway in human macrophages

© 1997 Federation of European Biochemical Societies.

Hepatic induction of cholesterol biosynthesis reflects a remote adaptive response to pneumococcal pneumonia

Community‐acquired pneumonia presents a spectrum of clinical phenotypes, from lobar pneumonia to septic shock, while mechanisms underlying progression are incompletely understood. In a transcriptomic and metabolomic study across tissues, we examined serotype‐specific regulation of signaling and metabolic pathways in C57BL/6 mice intratracheally instilled with either serotype 19F Streptococcus pneumoniae (S19; causing lobar pneumonia), or serotype 2 S. pneumoniae (S2; causing septic pneumococcal disease,) or vehicle (Todd‐Hewitt broth). Samples of lung, liver, and blood were collected at 6 and 24 h postinfection and subjected to microarray analysis and mass spectrometry. Results comprise a preferential induction of cholesterol biosynthesis in lobar pneumonia at low‐infection doses (105 colony forming units/mouse) leading to increased plasma cholesterol (vehicle: 1.8 ±0.12 mM, S2: 2.3±0.10 mM, S19: 2.9±0.15 mM; P>0.05, comparing S19 to vehicle and S2). This induction was pneumolysin dependent, as a pneumolysin‐deficient strain of serotype 19F failed to induce cholesterol biosynthesis (S19ΔPLY: 1.9±0.03 mM). Preincubation of pneumolysin with purified cholesterol or plasma from hypercholesterolemic mice prior to intratracheal instillation protected against lung barrier dysfunction and alveolar macrophage necrosis. Cholesterol may attenuate disease severity by neutralizing pneumolysin in the alveolar compartment and thus prevent septic disease progression.—Weber, M., Lambeck, S., Ding, N., Henken, S., Kohl, M., Deigner, H. P., Enot, D. P., Igwe, E. I., Frappart, L., Kiehntopf, M., Claus, R. A., Kamradt, T., Weih, D., Vodovotz, Y., Briles, D. E., Ogunniyi, A. D., Paton, J. C., Maus, U. A., Bauer, M. Hepatic induction of cholesterol biosynthesis reflects a remote adaptive response to pneumococcal pneumonia. FASEB J. 26, 2424‐2436 (2012). www.fasebj.org

Oxidized Low Density Lipoprotein Stimulates Protein Kinase C (PKC) Activity and Expression of PKC-Isotypes via Prostaglandin-H-Synthase in P388D1 Cells

Treatment of P388D1 macrophage-like cells with oxLDL enhanced protein kinase C (PKC) activity in cell extracts. Similar effects were induced by acetylated LDL (acLDL) and maleylated albumin (malBSA). Treatment with oxLDL, acLDL and malBSA was also accompanied by increased production of prostaglandins as well as by an enhanced level of prostaglandin H synthase 2 (cyclooxygenase 2, COX 2). Modified (lipo)proteins differentially affected the levels of individual cytosolic PKC-isoenzymes. Effects of oxLDL on PKC activity/expression were abrogated by indometacin, by pre-exposure to the dual lipoxygenase/cyclooxygenase inhibitor ML 3000 and by treatment with N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide (NS-398). These results suggest a predominantly COX 2-dependent and isotype-specific effect of modified (lipo)proteins on PKC.

Novel reversible, irreversible and fluorescent inhibitors of platelet-activating factor acetylhydrolase as mechanistic probes

Phosphatidylcholines (1-O-alcoxy-2-amino-2-desoxy-phosphocholines and 1-pyrene-labeled analogs) were synthesized and used to examine interactions with recombinant human PAF-acetylhydrolase (PAF-AH), an enzyme purified from plasma, and with macrophage-like U937 cells. Novel phosphatidylcholines containing a sn-2-carbamoylester group such as 1-O-hexadecyl-2-desoxy-2-amino-methylcarbamoyl-2-methyl-rac-glycer o-3-phosphocholine 11 were found to act as site-specific irreversible enzyme inhibitors with Ki-values up to 83 (K(irev)) and 177 (Ki(inact)) microm. The compounds exhibit only marginal inhibition of Ca2+-dependent phospholipases. Kinetic data show that phosphocholines carrying a terminal sn-1-pyrene moiety inhibit PAF-AH activity with an effectivity similar to analogs with an aliphatic chain. 1-O-Decyloxy-[10-(4-pyrenyl)-butoxy]-2-desoxy-2-amino-carbamoyl-me thyl-rac(-glycero-3-phosphocholine 13 could be used for enzyme labeling and to demonstrate an inhibitor-enzyme stoichiometry of 0.7:1. At 8 degrees C, the compound accumulated in the membranes of U937 cells, at 37 degrees C it was internalized into intracellular compartments. Structure activity studies in a mixed micelle assay indicated that the inhibition power of reversible and irreversible inhibitors increases along with the (sn)-1-chain length similar to the structure-dependent binding of ether phospholipids to the PAF-receptor. Unlike the situation at the (sn)-1-position, increasing chain length at the sn-2-position, or an alkyl branching of the glycerol backbone significantly reduced the inhibitory potency.

Prevalence of steric restrictions in enzymatic nitrile-hydrolysis of a preparation from Rhodococcus sp. 409

Abstract The size of the binding pocket of a nitrilase from Rhodococcus sp. 409 has been probed with 25 compounds and a basic active site model of potential predictive value has been established delineating the minimum pocket dimensions within a 4 A distance from the nitrile nitrogen atom. The total volume of this section of the model comprises 227.9 A 3 . Differential volume calculations were found to be indicative for hydrolysis and consistently, SYBYL CoMFA steric field reflects 70% of explained variance.

Multiplexing in Bioassays

Semiconductor nanoparticles, especially quantum dots (QDs), exhibit favourable optical properties for fluorescence imaging. Simultaneous excitation, without the need for monochromatic light and sharp emission bands allow the development of fast and sensitive multiplex immunoassays. Nano dyes can replace conventional organic dyes to increase the number of signals for multiplexing or be used in combination to form effective FRET-pairs. Advantageous properties like resistance to photobleaching or long fluorescence lifetimes at stable quantum yields and high extinction coefficients add to the benefits of quantum dots. Different strategies for data acquisition and experimental setup can improve conventional staining techniques to make them more sensitive, faster or versatile in multicolour imaging. The photostability allows long term light exposure of quantum dots, increasing the time frame for applications like live cell imaging. We provide a brief overview on current fluorescent tags and hardware suitable for multiplexing.