Butylated Hydroxytoluene (BHT) Protects SH-SY5Y Neuroblastoma Cells from Ferroptotic Cell Death: Insights from In Vitro and In Vivo Studies.

Ferroptosis is a special kind of programmed cell death that has been implicated in the pathogenesis of a large number of human diseases. It involves dysregulated intracellular iron metabolism and uncontrolled lipid peroxidation, which together initiate intracellular ferroptotic signalling pathways leading to cellular suicide. Pharmacological interference with ferroptotic signal transduction may prevent cell death, and thus patients suffering from ferroptosis-related diseases may benefit from such treatment. Butylated hydroxytoluene (BHT) is an effective anti-oxidant that is frequently used in oil chemistry and in cosmetics to prevent free-radical-mediated lipid peroxidation. Since it functions as a radical scavenger, it has previously been reported to interfere with ferroptotic signalling. Here, we show that BHT prevents RSL3- and ML162-induced ferroptotic cell death in cultured human neuroblastoma cells (SH-SY5Y) in a dose-dependent manner. It prevents the RSL3-induced oxidation of membrane lipids and normalises the RSL3-induced inhibition of the intracellular catalytic activity of glutathione peroxidase 4. The systemic application of BHT in a rat Alzheimer's disease model prevented the upregulation of the expression of ferroptosis-related genes. Taken together, these data indicate that BHT interferes with ferroptotic signalling in cultured neuroblastoma cells and may prevent ferroptotic cell death in an animal Alzheimer's disease model.

Different Structures-Similar Effect: Do Substituted 5-(4-Methoxyphenyl)-1H-indoles and 5-(4-Methoxyphenyl)-1H-imidazoles Represent a Common Pharmacophore for Substrate Selective Inhibition of Linoleate Oxygenase Activity of ALOX15?

Mammalian 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes that exhibit variable functionality in different cancer and inflammation models. The pathophysiological role of linoleic acid- and arachidonic acid-derived ALOX15 metabolites rendered this enzyme a target for pharmacological research. Several indole and imidazole derivatives inhibit the catalytic activity of rabbit ALOX15 in a substrate-specific manner, but the molecular basis for this allosteric inhibition remains unclear. Here, we attempt to define a common pharmacophore, which is critical for this allosteric inhibition. We found that substituted imidazoles induce weaker inhibitory effects when compared with the indole derivatives. In silico docking studies and molecular dynamics simulations using a dimeric allosteric enzyme model, in which the inhibitor occupies the substrate-binding pocket of one monomer, whereas the substrate fatty acid is bound at the catalytic center of another monomer within the ALOX15 dimer, indicated that chemical modification of the core pharmacophore alters the enzyme-inhibitor interactions, inducing a reduced inhibitory potency. In our dimeric ALOX15 model, the structural differences induced by inhibitor binding are translated to the hydrophobic dimerization cluster and affect the structures of enzyme-substrate complexes. These data are of particular importance since substrate-specific inhibition may contribute to elucidation of the putative roles of ALOX15 metabolites derived from different polyunsaturated fatty acids in mammalian pathophysiology.

Functional Characterization of Transgenic Mice Overexpressing Human 15-Lipoxygenase-1 (ALOX15) under the Control of the aP2 Promoter.

Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory, hyperproliferative, neurodegenerative, and metabolic diseases, but the physiological function of ALOX15 still remains a matter of discussion. To contribute to this discussion, we created transgenic mice (aP2-ALOX15 mice) expressing human ALOX15 under the control of the aP2 (adipocyte fatty acid binding protein 2) promoter, which directs expression of the transgene to mesenchymal cells. Fluorescence in situ hybridization and whole-genome sequencing indicated transgene insertion into the E1-2 region of chromosome 2. The transgene was highly expressed in adipocytes, bone marrow cells, and peritoneal macrophages, and ex vivo activity assays proved the catalytic activity of the transgenic enzyme. LC-MS/MS-based plasma oxylipidome analyses of the aP2-ALOX15 mice suggested in vivo activity of the transgenic enzyme. The aP2-ALOX15 mice were viable, could reproduce normally, and did not show major phenotypic alterations when compared with wildtype control animals. However, they exhibited gender-specific differences with wildtype controls when their body-weight kinetics were evaluated during adolescence and early adulthood. The aP2-ALOX15 mice characterized here can now be used for gain-of-function studies evaluating the biological role of ALOX15 in adipose tissue and hematopoietic cells.

IGFBP3 inhibits tumor growth and invasion of lung cancer cells and is associated with improved survival in lung cancer patients.

The insulin-like growth factor (IGF)-pathway is involved in tumor cell proliferation, metastasis, and survival. We aimed to find out what effects IGF binding protein 3 (IGFBP3) exerted on H1299 lung cancer (LC) cells in terms of tumor growth and invasion and whether IGFBP3 was associated with clinical and pathological parameters in a prospective cohort of LC patients. H1299 cells were transfected with an IGFBP3-expressing vector. Its influence on apoptosis induction via flow cytometry annexin V FITC assay, cell proliferation in 2D and 3D cell culture, and invasion were examined. Expression of several matrix metalloproteinases (MMPs) and inhibitors (TIMP-1) were also investigated in IGFBP3-transfected LC cells. Further, data on LC patients (n = 131), tumor characteristics, and survival were prospectively collected and correlated with IGFBP3 plasma levels. IGFBP3 did not influence apoptosis induction and 2D cell proliferation. However, both spheroid growth (3D proliferation) and invasion of IGFBP3-transfected cells planted in an extracellular matrix-based gel were significantly inhibited. IGFBP3 inhibited MMP-1 release, and the total MMP activity. In LC patients, higher IGFBP3 plasma levels correlated with both lower clinical tumor stage, grading, Ki-67 staining, and the absence of necrosis (P < 0.05, respectively). Increased IGFBP3 plasma levels were associated with improved overall survival (hazard ratio 0.37, P = 0.01). In conclusion, overexpressed IGFBP3 in a LC cell line inhibited tumor growth and invasion. Translating from bench to bedside, investigation of clinicopathological parameters confirmed these experimental results showing that higher IGFBP3 plasma levels were associated with less aggressive tumor growth, reduced tumor spread, and improved survival of LC patients.

Male guanine-rich RNA sequence binding factor 1 knockout mice (Grsf1-/-) gain less body weight during adolescence and adulthood.

The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the heterogenous nuclear ribonucleoprotein H/F (hnRNP H/F) family that binds to guanine-rich RNA sequences forming G-quadruplex structures. In mice and humans there are single copy GRSF1 genes, but multiple transcripts have been reported. GRSF1 has been implicated in a number of physiological processes (e.g. embryogenesis, erythropoiesis, redox homeostasis, RNA metabolism) but also in the pathogenesis of viral infections and hyperproliferative diseases. These postulated biological functions of GRSF1 originate from in vitro studies rather than complex in vivo systems. To assess the in vivo relevance of these findings, we created systemic Grsf1-/- knockout mice lacking exons 4 and 5 of the Grsf1 gene and compared the basic functional characteristics of these animals with those of wildtype controls. We found that Grsf1-deficient mice are viable, reproduce normally and have fully functional hematopoietic systems. Up to an age of 15 weeks they develop normally but when male individuals grow older, they gain significantly less body weight than wildtype controls in a gender-specific manner. Profiling Grsf1 mRNA expression in different mouse tissues we observed high concentrations in testis. Comparison of the testicular transcriptomes of Grsf1-/- mice and wildtype controls confirmed near complete knock-out of Grsf1 but otherwise subtle differences in transcript regulations. Comparative testicular proteome analyses suggested perturbed mitochondrial respiration in Grsf1-/- mice which may be related to compromised expression of complex I proteins. Here we present, for the first time, an in vivo complete Grsf1 knock-out mouse with comprehensive physiological, transcriptomic and proteomic characterization to improve our understanding of the GRSF1 beyond in vitro cell culture models.

Male Knock-in Mice Expressing an Arachidonic Acid Lipoxygenase 15B (Alox15B) with Humanized Reaction Specificity Are Prematurely Growth Arrested When Aging.

Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differentiation and in the pathogenesis of inflammation. The mouse genome involves seven functional Alox genes and the encoded enzymes share a high degree of amino acid conservation with their human orthologs. There are, however, functional differences between mouse and human ALOX orthologs. Human ALOX15B oxygenates arachidonic acid exclusively to its 15-hydroperoxy derivative (15S-HpETE), whereas 8S-HpETE is dominantly formed by mouse Alox15b. The structural basis for this functional difference has been explored and in vitro mutagenesis humanized the reaction specificity of the mouse enzyme. To explore whether this mutagenesis strategy may also humanize the reaction specificity of mouse Alox15b in vivo, we created Alox15b knock-in mice expressing the arachidonic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and develop normally up to an age of 24 weeks. At later developmental stages, male Alox15b-KI mice gain significantly less body weight than outbred wildtype controls, but this effect was not observed for female individuals. To explore the possible reasons for the observed gender-specific growth arrest, we determined the basic hematological parameters and found that aged male Alox15b-KI mice exhibited significantly attenuated red blood cell parameters (erythrocyte counts, hematocrit, hemoglobin). Here again, these differences were not observed in female individuals. These data suggest that humanization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic system in males, which is paralleled by a premature growth arrest.

Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis.

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.

Dichloroacetate and PX-478 exhibit strong synergistic effects in a various number of cancer cell lines.

BACKGROUND: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms. METHODS: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells. RESULTS: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment. CONCLUSIONS: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.

New insight into the role of glutathione reductase in glutathione peroxidase-like activity determination by coupled reductase assay: Molecular Docking Study.

Previously we have shown that among 15 substituted salicyloyl (2-hydroxybenzoyl) 5-seleninic acids (SSAs) 4 compounds with longer side chains or a cyclohexyl group exhibit no glutathione peroxidase (GPx)-like activity in the coupled reductase assay. Experimental inhibition of glutathione reductase (GR) by the selenenylsulfide (a main intermediate in the catalytic cycle for GPx-like activity determination) of one of the inactive compounds led us to assess the interactions between 15 selenenylsulfide compounds and the active site of GR by molecular docking. Docking results showed that S and Se atoms in selenenylsulfides of the compounds with no GPx-like activity were beyond 5 Šfrom S atom of Cys-58 or N atom of imidazole ring of His-467 (Root Mean Square Distances for general assessment of 3 major distances were over 4.8 Å) in the active site, so that they could not be catalyzed to be reduced by GR. Furthermore, their docking scores over 89 Kcal/mol meant that the selenenylsulfides were bound too strongly to the active site to leave it, leading eventually to inhibition of GR. We also applied the molecular docking to other GPx mimics such as ebselen, cyclic seleninate esters and di(propylaminomethylphenyl) diselenides to explain the differences in their GPx-like activity depending to the assays used. Our results suggest that the reduction of a selenenylsulfide by GR plays a positive role in GPx-like activity of GPx mimics in the coupled assay and recommended the prediction of possibility and strength of GPx-like activity by molecular docking before entering experimental research.

Specific overexpression of 15-lipoxygenase in endothelial cells promotes cancer cell death in an in vivo Lewis lung carcinoma mouse model.

PURPOSE: Lipoxygenases (LOX) have been implicated in carcinogenesis, however both pro- and anti-carcinogenic effects have been reported in different cancer models. Using transgenic mice, which specifically overexpress human 15-lipoxygenase (ALOX15) in endothelial cells (EC), we previously demonstrated significant inhibition of tumor development. In the Lewis lung carcinoma (LLC) model, the primary tumor developed similarly in both wild type (WT) and ALOX15 overexpressing mice. However, metastases development was significantly inhibited in the transgenic mice. Here, we explored the molecular basis for the anti-metastatic effect of endothelial cell specific ALOX15 overexpression. MATERIALS/METHODS: We used ALOX15 overexpressing mice, and in-vitro cell model to evaluate the molecular effect of ALOX15 on EC and LLC cells. RESULTS: When LLC cells were injected in WT and ALOX15 overexpressing mice, we observed a higher degree of apoptosis and necrosis in primary and metastatic tumors of ALOX15 overexpressing animals. These anti-carcinogenic and anti-metastatic effects were paralleled by augmented expression of cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) and of the peroxisome proliferators-activated receptor (PPAR)γ and by downregulation of the steady state concentrations of connexin26 mRNA. Consistent with these in vivo effects, ALOX15 overexpression in LLC and HeLa cancer cells in vitro significantly reduced cell viability in culture. In contrast, similar treatment of non-cancerous B2B epithelial cells did not impact cell viability. CONCLUSIONS: Taken together, our data suggests that endothelial cell specific overexpression of ALOX15 promotes apoptosis and necrosis in primary and metastatic tumors in mice, by upregulation of P21 and PPARγ expression in adjacent cancer cells.

Do lipoxygenases occur in viruses?: Expression and characterization of a viral lipoxygenase-like protein did not provide evidence for the existence of functional viral lipoxygenases.

Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and animals. In bacteria, these enzymes are rare and have been introduced via horizontal gene transfer. Since viruses function as horizontal gene transfer vectors and since lipoxygenases may be helpful for releasing assembled virus particles from host cells we explored whether these enzymes may actually occur in viruses. For this purpose we developed a four-step in silico screening strategy and searching the publically available viral genomes for lipoxygenase-like sequences we detected a single functional gene in the genome of a mimivirus infecting Acantamoeba polyphaga. The primary structure of this protein involved two putative metal ligand clusters but the recombinant enzyme did neither contain iron nor manganese. Most importantly, it did not exhibit lipoxygenase activity. These data suggests that this viral lipoxygenase-like sequence does not encode a functional lipoxygenase and that these enzymes do not occur in viruses.

Crystal structure and functional characterization of selenocysteine-containing glutathione peroxidase 4 suggests an alternative mechanism of peroxide reduction.

Glutathione peroxidases (GPX) are anti-oxidative enzymes that reduce organic and inorganic hydroperoxides to the corresponding alcohols at the expense of reduced glutathione. The human genome involves eight GPX genes and five of them encode for selenocysteine-containing enzymes. Among the human GPX-isoforms, GPX4 is unique since it is capable of reducing complex hydroperoxy ester lipids such as hydroperoxy phospholipids and hydroperoxy cholesterolesters. Using a number of genetically modified mouse strains the biological role of GPX4 has comprehensively characterized but the molecular enzymology is less well explored. This lack of knowledge is partly related to the fact that mammalian selenoproteins are not high-level expressed in conventional overexpression systems. To explore the structural and functional properties of human GPX4 we expressed this selenoprotein in a cysteine-auxotrophic E. coli strain using a semi-chemical expression strategy. The recombinant enzyme was purified in mg amounts from the bacterial lysate to electrophoretic homogeneity and characterized with respect to its protein-chemical and enzymatic properties. Its crystal structure was solved at 1.3 Šresolution and the X-ray data indicated a monomeric protein, which contains the catalytic selenium at the redox level of the seleninic acid. These data suggest an alternative reaction mechanism involving three different redox states (selenol, selenenic acid, seleninic acid) of the catalytically active selenocysteine.

Structural and functional basis of phospholipid oxygenase activity of bacterial lipoxygenase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa expresses a secreted LOX-isoform (PA-LOX, LoxA) capable of oxidizing polyenoic fatty acids to hydroperoxy derivatives. Here we report high-level expression of this enzyme in E. coli and its structural and functional characterization. Recombinant PA-LOX oxygenates polyenoic fatty acids including eicosapentaenoic acid and docosahexaenoic acid to the corresponding (n-6)S-hydroperoxy derivatives. This reaction involves abstraction of the proS-hydrogen from the n-8 bisallylic methylene. PA-LOX lacks major leukotriene synthase activity but converts 5S-HETE and 5S,6R/S-DiHETE to anti-inflammatory and pro-resolving lipoxins. It also exhibits phospholipid oxygenase activity as indicated by the formation of a specific pattern of oxygenation products from different phospholipid subspecies. Multiple mutagenesis studies revealed that PA-LOX does not follow classical concepts explaining the reaction specificity of mammalian LOXs. The crystal structure of PA-LOX was solved with resolutions of up to 1.48Å and its polypeptide chain is folded as single domain. The substrate-binding pocket consists of two fatty acid binding subcavities and lobby. Subcavity-1 contains the catalytic non-heme iron. A phosphatidylethanolamine molecule occupies the substrate-binding pocket and its sn1 fatty acid is located close to the catalytic non-heme iron. His377, His382, His555, Asn559 and the C-terminal Ile685 function as direct iron ligands and a water molecule (hydroxyl) completes the octahedral ligand sphere. Although the biological relevance of PA-LOX is still unknown its functional characteristics (lipoxin synthase activity) implicate this enzyme in a bacterial evasion strategy aimed at downregulating the hosts' immune system.

Mutagenesis of triad determinants of rat Alox15 alters the specificity of fatty acid and phospholipid oxygenation.

Among lipoxygenases ALOX15 orthologs are somewhat peculiar because of their capability of oxygenating polyenoic fatty acids even if they are incorporated in complex lipid-protein assemblies. ALOX15 orthologs of different species have been characterized before, but little is known about the corresponding rat enzyme. Since rats are frequently employed as models in biomedical research we expressed rat Alox15 as recombinant protein in pro- and eukaryotic expression systems and characterized the enzyme with respect to its enzymatic properties. The enzyme oxygenated free arachidonic acid mainly to 12S-HpETE with 15S-HpETE only contributing 10% to the product mixture. Multiple directed mutagenesis studies indicated applicability of the triad concept with particular importance of Leu353 and Ile593 as specificity determinants. Ala404Gly exchange induced subtle alterations in enantioselectivity suggesting partial applicability of the Coffa/Brash concept. Wildtype rat Alox15 and its 15-lipoxygenating Leu353Phe mutant are capable of oxygenating ester lipids of biomembranes and high-density lipoproteins. For the wildtype enzyme 13S-HODE and 12S-HETE were identified as major oxygenation products but for the Leu353Phe mutant 13S-HODE and 15S-HETE prevailed. These data indicate for the first time that mutagenesis of triad determinants modifies the reaction specificity of ALOX15 orthologs with free fatty acids and complex ester lipids in a similar way.

Mammalian lipoxygenases and their biological relevance.

Lipoxygenases (LOXs) form a heterogeneous class of lipid peroxidizing enzymes, which have been implicated not only in cell proliferation and differentiation but also in the pathogenesis of various diseases with major public health relevance. As other fatty acid dioxygenases LOXs oxidize polyunsaturated fatty acids to their corresponding hydroperoxy derivatives, which are further transformed to bioactive lipid mediators (eicosanoids and related substances). On the other hand, lipoxygenases are key players in the regulation of the cellular redox homeostasis, which is an important element in gene expression regulation. Although the first mammalian lipoxygenases were discovered 40 years ago and although the enzymes have been well characterized with respect to their structural and functional properties the biological roles of the different lipoxygenase isoforms are not completely understood. This review is aimed at summarizing the current knowledge on the physiological roles of different mammalian LOX-isoforms and their patho-physiological function in inflammatory, metabolic, hyperproliferative, neurodegenerative and infectious disorders. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling.

Leukotrienes are pro-inflammatory lipid mediators, which are biosynthesized via the lipoxygenase pathway of the arachidonic acid cascade. Lipoxygenases form a family of lipid peroxidizing enzymes and human lipoxygenase isoforms have been implicated in the pathogenesis of inflammatory, hyperproliferative (cancer) and neurodegenerative diseases. Lipoxygenases are not restricted to humans but also occur in a large number of pro- and eucaryotic organisms. Lipoxygenase-like sequences have been identified in the three domains of life (bacteria, archaea, eucarya) but because of lacking functional data the occurrence of catalytically active lipoxygenases in archaea still remains an open question. Although the physiological and/or pathophysiological functions of various lipoxygenase isoforms have been studied throughout the last three decades there is no unifying concept for the biological importance of these enzymes. In this review we are summarizing the current knowledge on the distribution of lipoxygenases in living single and multicellular organisms with particular emphasis to higher vertebrates and will also focus on the genetic diversity of enzymes and receptors involved in human leukotriene signaling.

Differential expression of secretoglobins in normal ovary and in ovarian carcinoma--overexpression of mammaglobin-1 is linked to tumor progression.

Secretoglobins (SCGB), such as mammaglobin 1 (MGB1, SCGB2A2), mammaglobin 2 (MGB2, SCGB2A1) and lipophilin B (LIPB, SCGB1D2), have been related to carcinogenesis. We profiled expression of MGB1, MGB2 and LIPB in human tissues and ovarian carcinoma and explored the impact of SCGB overexpression on cell proliferation. MGB1, MGB2 and LIPB mRNA are expressed at variable levels in most human tissues and we observed significant bilateral correlations between the different secretoglobins. Concerted overexpression of MGB1 and LIPB resulted in significant increase in cell proliferation. In clinical specimens of ovarian carcinoma we measured elevated concentrations of secretoglobin mRNA and for MGB1 this up-regulation was confirmed on the protein level. Overexpression of MGB1 positively correlated with the FIGO stage, the tumor grade and the mitotic index suggesting a patho-physiological role of the protein. Our data indicate that MGB1, MGB2 and LIPB mRNAs are expressed at low levels in human tissues but basal expression is upregulated in ovarian cancer. The in vivo correlation between nuclear MGB1 localization and the mitotic rate in ovarian cancer as well as the increased cell proliferation induced by secretoglobin overexpression in ovarian cancer cell lines suggest a pathophysiological role of these proteins in ovarian cancer.

Interaction of cyclic mechanical stretch and toll-like receptor 4-mediated innate immunity in rat alveolar type II cells.

BACKGROUND AND OBJECTIVE: In cases of infection-induced acute lung injury, mechanical ventilation might be necessary to maintain oxygenation. Although low tidal volume ventilation is applied, alveolar over-distension may occur and result in ventilator-induced lung injury. In this study, we investigate (i) the influence of lipopolysaccharide (LPS) stimulation on high-amplitude stretching; and (ii) the effect of stretching on LPS-mediated immune response in isolated rat alveolar type II cells. METHODS: Type II cells were incubated with LPS and stretched for 24 h on elastic membranes. Initially we examined apoptosis and lactic acid dehydrogenase release in LPS-treated stretched cells. Furthermore we determined toll-like receptor (TLR) 4 expression, TLR4 signalling by analysis of nuclear factor κB (NF-κB) activation and the secretion of inflammatory cytokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-2, interleukin-1 beta, tumour necrosis factor alpha). RESULTS: Our results show that LPS increases apoptosis and cytotoxicity in high amplitude stretched cells. Stretching and LPS activate NF-κB. The LPS influence is the prevailing one while no synergistic effects were observed by additional stretching. LPS stimulates an increased secretion of the inflammatory mediators only. Stretching had no influence on cytokines secretion. CONCLUSIONS: We conclude that activation of TLR4 mediated immunity intensifies cell damage caused by stretching whereas in return stretching had no influence on TLR4 mediated innate immunity.

The N-terminal ß-barrel domain of mammalian lipoxygenases including mouse 5-lipoxygenase is not essential for catalytic activity and membrane binding but exhibits regulatory functions.

Mammalian lipoxygenases (LOXs) have been implicated in cell differentiation and in the pathogenesis of inflammatory and hyperproliferative diseases. The available structural information indicated that lipoxygenases constitute single polypeptide chain enzymes consisting of a small N-terminal ß-barrel domain and a larger C-terminal subunit that harbors the catalytic non-heme iron. Because of its structural similarity to C2-domains of lipases the N-terminal ß-barrel domain of lipoxygenases, which comprises about 110 amino acids, has been implicated in membrane binding and activity regulation. To explore the functional relevance of the C2-domain in more detail and to develop a more comprehensive hypothesis on the biological role of this structural subunit we performed gene technical truncation on various mammalian LOX isoforms (12/15-LOXs of various species, human 15-LOX2, mouse 5-LOX) and quantified catalytic activity and membrane binding properties of the truncated recombinant enzyme species. We found that the C2-domain is not essential for catalytic activity and does hardly impact reaction specificity. Truncated enzyme species exhibit impaired membrane binding properties and altered reaction kinetics. Taken together, our data suggests a regulatory importance of the N-terminal ß-barrel domain for mammalian lipoxygenase isoforms.