Inhibition and deficiency of the immunoproteasome subunit LMP7 attenuates LCMV-induced meningitis

In addition to antigen processing, immunoproteasomes were recently shown to exert functions influencing cytokine production by monocytes and T cells, T-helper cell differentiation, and T-cell survival. Moreover, selective inhibition of the immunoproteasome subunit LMP7 ameliorated symptoms of autoimmune diseases including CD4(+) T-cell mediated EAE. In this study, we show that LMP7 also plays a crucial role in the pathogenesis of lymphocytic choriomeningitis virus (LCMV)-induced meningitis mediated by CTLs. Mice lacking functional LMP7 display delayed and reduced clinical signs of disease accompanied by a strongly decreased inflammatory infiltration into the brain. Interestingly, we found that selective inhibition and genetic deficiency of LMP7 affect the pathogenesis of LCMV-induced meningitis in a distinct manner. Our findings support the important role of LMP7 in inflammatory disorders and suggest immunoproteasome inhibition as a novel strategy against inflammation-induced neuropathology in the CNS.

Inhibition of glutathione synthesis reduces chilling tolerance in maize

The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine (gamma EC) synthetase, the first enzyme of GSH synthesis. At 25 degrees C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 degrees C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or gamma EC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH. which also induced a decrease in GR activity.

Organ inflammation in porcine Escherichia coli sepsis is markedly attenuated by combined inhibition of C5 and CD14.

Sepsis is an infection-induced systemic inflammatory syndrome, potentially causing organ failure. We previously showed attenuating effects on inflammation, thrombogenicity and haemodynamics by inhibiting the Toll-like receptor co-factor CD14 and complement factor C5 in a porcine Escherichia coli-induced sepsis model. The present study explored the effect on organ inflammation in these pigs. Tissue samples were examined from the combined treatment group (n = 8), the positive (n = 8) and negative (n = 6) control groups after 4h of sepsis. Inflammatory biomarkers were measured using ELISA, multiplex and qPCR analysis. Combined inhibition of C5 and CD14 markedly attenuated IL-1β by 31-66% (P < 0.05) and IL-6 by 54-96% (P < 0.01) in liver, kidney, lung and spleen; IL-8 by 65-100% in kidney, lung, spleen, and heart (P < 0.05) and MCP-1 by 46-69% in liver, kidney, spleen and heart (P < 0.05). Combined inhibition significantly attenuated tissue factor mRNA upregulation in spleen (P < 0.05) and IP-10 mRNA upregulation in four out of five organs. Finally, C5aR mRNA downregulation was prevented in heart and kidney (P < 0.05). Combined inhibition of C5 and CD14 thus markedly attenuated inflammatory responses in all organs examined. The anti-inflammatory effects observed in lung and heart may explain the delayed haemodynamic disturbances observed in septic pigs receiving combined inhibition of C5 and CD14.

Optimized on-line enantioselective capillary electrophoretic method for kinetic and inhibition studies of drug metabolism mediated by cytochrome P450 enzymes.

Pharmacokinetic and pharmacodynamic properties of a chiral drug can significantly differ between application of the racemate and single enantiomers. During drug development, the characteristics of candidate compounds have to be assessed prior to clinical testing. Since biotransformation significantly influences drug actions in an organism, metabolism studies represent a crucial part of such tests. Hence, an optimized and economical capillary electrophoretic method for on-line studies of the enantioselective drug metabolism mediated by cytochrome P450 enzymes was developed. It comprises a diffusion-based procedure, which enables mixing of the enzyme with virtually any compound inside the nanoliter-scale capillary reactor and without the need of additional optimization of mixing conditions. For CYP3A4, ketamine as probe substrate and highly sulfated γ-cyclodextrin as chiral selector, improved separation conditions for ketamine and norketamine enantiomers compared to a previously published electrophoretically mediated microanalysis method were elucidated. The new approach was thoroughly validated for the CYP3A4-mediated N-demethylation pathway of ketamine and applied to the determination of its kinetic parameters and the inhibition characteristics in presence of ketoconazole and dexmedetomidine. The determined parameters were found to be comparable to literature data obtained with different techniques. The presented method constitutes a miniaturized and cost-effective tool, which should be suitable for the assessment of the stereoselective aspects of kinetic and inhibition studies of cytochrome P450-mediated metabolic steps within early stages of the development of a new drug.

SrGAP2-Dependent Integration of Membrane Geometry and Slit-Robo-Repulsive Cues Regulates Fibroblast Contact Inhibition of Locomotion

Migrating fibroblasts undergo contact inhibition of locomotion (CIL), a process that was discovered five decades ago and still is not fully understood at the molecular level. We identify the Slit2-Robo4-srGAP2 signaling network as a key regulator of CIL in fibroblasts. CIL involves highly dynamic contact protrusions with a specialized actin cytoskeleton that stochastically explore cell-cell overlaps between colliding fibroblasts. A membrane curvature-sensing F-BAR domain pre-localizes srGAP2 to protruding edges and terminates their extension phase in response to cell collision. A FRET-based biosensor reveals that Rac1 activity is focused in a band at the tip of contact protrusions, in contrast to the broad activation gradient in contact-free protrusions. SrGAP2 specifically controls the duration of Rac1 activity in contact protrusions, but not in contact-free protrusions. We propose that srGAP2 integrates cell edge curvature and Slit-Robo-mediated repulsive cues to fine-tune Rac1 activation dynamics in contact protrusions to spatiotemporally coordinate CIL.

Inhibition of apoptosis by intracellular protozoan parasites

Protozoan parasites which reside inside a host cell avoid direct destruction by the immune system of the host. The infected cell, however, still has the capacity to counteract the invasive pathogen by initiating its own death, a process which is called programmed cell death or apoptosis. Apoptotic cells are recognised and phagocytosed by macrophages and the parasite is potentially eliminated together with the infected cell. This potent defence mechanism of the host cell puts strong selective pressure on the parasites which have, in turn, evolved strategies to modulate the apoptotic program of the host cell to their favour. Within the last decade, the existence of cellular signalling pathways which inhibit the apoptotic machinery has been demonstrated. It is not surprising that intracellular pathogens subvert these pathways to ensure their own survival in the infected cell. Molecular mechanisms which interfere with apoptotic pathways have been studied extensively for viruses and parasitic bacteria, but protozoan parasites have come into focus only recently. Intracellular protozoan parasites which have been reported to inhibit the apoptotic program of the host cell, are Toxoplasma gondii, Trypanosoma cruzi, Leishmania sp., Theileria sp., Cryptosporidium parvum, and the microsporidian Nosema algerae. Although these parasites differ in their mechanism of host cell entry and in their final intracellular localisation, they might activate similar pathways in their host cells to inhibit apoptosis. In this respect, two families of molecules, which are known for their capacity to interrupt the apoptotic program, are currently discussed in the literature. First, the expression of heat shock proteins is often induced upon parasite infection and can directly interfere with molecules of the cellular death machinery. Secondly, a more indirect effect is attributed to the parasite-dependent activation of NF-kappaB, a transcription factor that regulates the transcription of anti-apoptotic molecules.

The inhibition of NF-kappaB activation pathways and the induction of apoptosis by dithiocarbamates in T cells are blocked by the glutathione precursor N-acetyl-L-cysteine

Nuclear factor-kappaB regulates genes that control immune and inflammatory responses and are involved in the pathogenesis of several diseases, including AIDS and cancer. It has been proposed that reactive oxygen intermediates participate in NF-kappaB activation pathways, and compounds with putative antioxidant activity such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) have been used interchangeably to demonstrate this point. We examined their effects, separately and combined, on different stages of the NF-kappaB activation pathway, in primary and in transformed T cells. We show that NAC, contrary to its reported role as an NF-kappaB inhibitor, can actually enhance rather than inhibit IkappaB degradation and, most importantly, show that in all cases NAC exerts a dominant antagonistic effect on PDTC-mediated NF-kappaB inhibition. This was observed at the level of IkappaB degradation, NF-kappaB DNA binding, and HIV-LTR-driven reporter gene expression. NAC also counteracted growth arrest and apoptosis induced by dithiocarbamates. Antagonistic effects were further observed at the level of jun-NH2-terminal kinase, p38 and ATF-2 activation. Our findings argue against the widely accepted assumption that NAC inhibits all NF-kappaB activation pathways and shows that two compounds, previously thought to function through a common inhibitory mechanism, can also have antagonistic effects.

MECHANISMS OF SPECIES DIFFERENCES IN BRAIN ACETYLCHOLINESTERASE SENSITIVITY TO ORGANOPHOSPHATE INHIBITION

In vitro incubation of acetylcholinesterase from brain tissue of several species with organophosphate compounds indicated that the concentrations required to inhibit 50% of acetylcholinesterase activity (IC(,50)) differed from species to species for the same compound (Murphy, et al., 1968; Andersen, et al., 1972, 1977 and 1978).^ The hypothesis that non-specific binding proteins (Lauwerys and Murphy, 1969a,b) exerts a protective effect on acetylcholinesterase, and thus cause the differences observed in IC(,50) studies was tested by a ('3)H-DFP binding experiment. It was found that differences in the amount of non-specific binding protein cannot explain the observed differences observed in IC(,50) studies.^ An alternative hypothesis, that acetylcholinesterase from different species have different affinities for binding and/or different rates of phosphorylation by organophosphate insecticides was tested by determining the apparent affinity constant (k(,a)) and apparent rate of phosphorylation (k(,p)). Kinetic studies indicated that acetylcholinesterases from different species have different sensitivities to inhibition by organophosphate insecticides, and the differences are due to different affinities for binding and/or different rates of phosphorylation by the same organophosphate compound.^ Studies of the spontaneous reactivation of acetylcholinesterase after inhibition by organophosphate insecticides also indicated that acetylcholinesterases from different species have different rates and extents of spontaneous reactivation. This further substantiates the hypothesis that acetylcholinesterases from different species have different kinetic characteristics with respect to organophosphate insecticides inhibition.^ Eleven paraoxon analogs were synthesized for a quantitative structure-activity relationship study. It was found that the electron-withdrawing power ((sigma)) and hydrophobicity ((PARAGR)) of the substituent are important in determining the anti-cholinesterase activity of paraoxon analogs. Thus, predictions of species differences in acetylcholinesterase sensitivities to paraoxon analogs can be made if the physicochemical parameters ((sigma) and (PARAGR)) of the substituents are known.^ In another approach, i.e. enzyme modeling, the sensitivity of rat brain acetylcholinesterase to organophosphate insecticides was used as the independent variable to predict the sensitivities of acetylcholinesterases from other species to the same compound. Regression equations were derived for each species based on nineteen organophosphate insecticides studied. It was found, that in addition to paraoxon analogs, this method is also applicable to other organophosphate compounds with wide variations in structure. Thus, the sensitivities of acetylcholinesterases from other species can also be predicted from the sensitivity of rat brain acetylcholinesterase. ^

INHIBITION OF THE MITOCHONDRIAL RESPIRATION OF TUMOR CELLS BY SOLUBLE FACTORS RELEASED BY ACTIVATED MACROPHAGES (CYTOTOXICITY, ELECTRON TRANSPORT, MONOCYTE)

Particular interest has been directed towards the macrophage as a primary antineoplastic cell due to its tumoricidal properties in vitro and the observation that an inverse relationship exists between the number of macrophages infiltrating a tumor and metastatic potential. The mechanism of macrophage-mediated injury of tumor cells remains unknown. Recently, it has been shown that injured tumor cells have defective mitochondrial respiration. Our studies have shown that activated macrophages can release soluble factors which can alter tumor cell respiration.^ The effects of a conditioned supernatant (CS) from cultures of activated macrophages on tumor cell (TC) mitochondrial respiration was studied. CS was obtained by incubation of BCG-elicited, murine peritoneal macrophage with RPMI-1640 supplemented with 10% FCS and 50 ng/ml bacterial endotoxin. This CS was used to treat cultures of EMT-6 TC for 24 hours. Mitochondrial respiration was measured polarigraphically using a Clark-type oxygen electrode. Cell growth rate was assessed by ('3)H-Thymidine incorporation. Exposure of EMT-6 TC to CS resulted in the inhibition of malate and succinate oxidation 76.6% and 72.9%, respectively. While cytochrome oxidase activity was decreased 61.1%. This inhibition was accompanied by a 98.8% inhibition of DNA synthesis (('3)H-Thymidine incorporation). Inhibition was dose-related with a 21.3% inhibition of succinate oxidase from a 0.3 ml dose of CS and a 50% inhibition with 1.0 mls. Chromatography of CS on Sephacryl S-200 resulted in isolation of an 80,000 and a 55,000 dalton component which contained the respiration inhibiting activity (RIF). These factors were distinct from a 120,000 dalton cytolytic factor determined by bioassay on Actinomycin-D treated L929 cells. RIF activity was also distinct from several other cytostatic factors but was itself associated with 2 peaks of cytostatic activity. Characterization of the RIF activity showed that it was destroyed by trypsin and heat (100(DEGREES)C, 5 min). It was stable over a broad range of pH (4-9) and its production was inhibited by cycloheximide. The RIF did not have a direct effect on isolated mitochondria of TC nor did it induce the formation of a stable intracellular toxin for mitochondria.^ In conclusion, activated macrophages synthesize and secrete an 80,000 and a 55,000 dalton protein which inhibits the mitochondrial metabolism of TC. These factors induce a cytostatic but not a cytolytic effect on TC.^ The macrophage plays a role in the control of normal and tumor cell growth and in tissue involution. Inhibition of respiration may be one mechanism used by macrophages to control cell growth.^

Mechanism of damage sensing and cell killing following the inhibition of nucleotide excision repair by fludarabine in quiescent cells

Inhibition of DNA repair by the nucleoside of fludarabine (F-ara-A) induces toxicity in quiescent human cells. The sensing and signaling mechanisms following DNA repair inhibition by F-ara-A are unknown. The central hypothesis of this project was that the mechanistic interaction of a DNA repair initiating agent and a nucleoside analog initiates an apoptotic signal in quiescent cells. The purpose of this research was to identify the sensing and signaling mechanism(s) that respond to DNA repair inhibition by F-ara-A. Lymphocytes were treated with F-ara-A, to accumulate the active triphosphate metabolite and subsequently DNA repair was activated by UV irradiation. Pre-incubation of lymphocytes with 3 μM F-ara-A inhibited DNA repair initiated by 2 J/m2 UV and induced greater than additive apoptosis after 24 h. Blocking the incorporation of F-ara-A nucleotide into repairing DNA using 30 μM aphidicolin considerably lowered the apoptotic response. ^ Wild-type quiescent cells showed a significant loss in viability than did cells lacking functional sensor kinase DNA-PKcs or p53 as measured by colony formation assays. The functional status of ATM did not appear to affect the apoptotic outcome. Immunoprecipitation studies showed an interaction between the catalytic sub-unit of DNA-PK and p53 following DNA repair inhibition. Confocal fluorescence microscopy studies have indicated the localization pattern of p53, DNA-PK and γ-H2AX in the nucleus following DNA damage. Foci formation by γ-H2AX was seen as an early event that is followed by interaction with DNA-PKcs. p53 serine-15 phosphorylation and accumulation were detected 2 h after treatment. Fas/Fas ligand expression increased significantly after repair inhibition and was dependent on the functional status of p53. Blocking the interaction between Fas and Fas ligand by neutralizing antibodies significantly rescued the apoptotic fraction of cells. ^ Collectively, these results suggest that incorporation of the nucleoside analog into repair patches is critical for cytotoxicity and that the DNA damage, while being sensed by DNA-PK, may induce apoptosis by a p53-mediated signaling mechanism. Based on the results, a model is proposed for the sensing of F-ara-A-induced DNA damage that includes γ-H2AX, DNA-PKcs, and p53. Targeting the cellular DNA repair mechanism can be a potential means of producing cytotoxicity in a quiescent population of neoplastic cells. These results also provide mechanistic support for the success of nucleoside analogs with cyclophosphamide or other agents that initiate excision repair processes, in the clinic. ^