5-lipoxygenase Protein Research Articles

Identification of Active Compound from Mitragyna speciosa Leave as Antiinflammation Agent: In Silico Study.

The study aims to identify the most responsible compound for the antiinflammation activity from Mitragyna speciosa leaves. Seventeen compounds previously reported to have been isolated from the leave were virtually screened against human 5-lipoxygenase protein and analyzed according to their binding energies. The native ligand used was arachidonic acid, and mitragynine was found to be the strongest binding compound (Pubchem ID: 3034396). In addition, ADMET profiling shows that mitragynine was not violating Lipinski’s rule of five and was not toxic.

Neuroprotective effect of peanut against oxidative stress in streptozotocin-induced diabetic rats

ABSTRACT Diabetes mellitus is characterized by structural abnormalities, oxidative stress and neuroinflammation. This study aimed to determine the antioxidative therapeutic effects of peanut supplementation in improving brain damage resulted from streptozotocin (STZ)-induced diabetes. Forty male Wistar albino rats (Rattus novergicus) were categorized into four groups: control, peanut-supplemented, diabetic, diabetic and peanut-supplemented. The brain was dissected and subjected to biochemical analyses which indicated the role of diabetes in downregulating the expression of superoxide dismutase (SOD), dopamine (DA), serotonin (5-HT), adenosine triphosphate (ATP) and upregulating the levels of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), 5-lipoxygenase (5-lOX), 8-hydroxy-deoxy guanosine (8-OHdG), Amyloid-β, α-amylase and tau protein expression levels. peanut treatment enhanced the diabetic-dependent brain histopathological features in the cerebrum and hippocampus, the immunohistochemical localization indicated significant downregulation at the expression of synaptophysin and caspase 3. Peanut supplementation significantly downregulated the gene expression of BAX, SOD, Myloperoxidase (MPO), Tumor necrosis factor- α (TNF-α), peroxisome proliferator-activated receptors (PPAR-α and PPAR-γ) and upregulated glial fibrillary acidic protein (GFAP) expression. In conclusion, peanut supplementation showed therapeutic potential against brain damage induced by diabetes. Peanut treatment significantly protected the brain from diabetic-related oxidative stress, and increased dopamine and serotonin levels by restoring the redox balance.

Palladium(II) Complexes of Substituted Salicylaldehydes: Synthesis, Characterization and Investigation of Their Biological Profile.

Five palladium(II) complexes of substituted salicylaldehydes (X-saloH, X = 4-Et2N (for 1), 3,5-diBr (for 2), 3,5-diCl (for 3), 5-F (for 4) or 4-OMe (for 5)) bearing the general formula [Pd(X-salo)2] were synthesized and structurally characterized. The crystal structure of complex [Pd(4-Et2N-salo)2] was determined by single-crystal X-ray crystallography. The complexes can scavenge 1,1-diphenyl-picrylhydrazyl and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radicals and reduce H2O2. They are active against two Gram-positive (Staphylococcus aureus and Bacillus subtilis) and two Gram-negative (Escherichia coli and Xanthomonas campestris) bacterial strains. The complexes interact strongly with calf-thymus DNA via intercalation, as deduced by diverse techniques and via the determination of their binding constants. Complexes interact reversibly with bovine and human serum albumin. Complementary insights into their possible mechanisms of bioactivity at the molecular level were provided by molecular docking calculations, exploring in silico their ability to bind to calf-thymus DNA, Escherichia coli and Staphylococcus aureus DNA-gyrase, 5-lipoxygenase, and membrane transport lipid protein 5-lipoxygenase-activating protein, contributing to the understanding of the role complexes 1–5 can play both as antioxidant and antibacterial agents. Furthermore, in silico predictive tools have been employed to study the chemical reactivity, molecular properties and drug-likeness of the complexes, and also the drug-induced changes of gene expression profile (as protein- and mRNA-based prediction results), the sites of metabolism, the substrate/metabolite specificity, the cytotoxicity for cancer and non-cancer cell lines, the acute rat toxicity, the rodent organ-specific carcinogenicity, the anti-target interaction profiles, the environmental ecotoxicity, and finally the activity spectra profile of the compounds.

Loss of detection of fatty acid-metabolizing proteins in Western blot analyses – Impact of sample heating

When performing western blots for protein detection using the classical Laemmli method, experimenters often encounter difficulties with the detection of transmembrane proteins involved in lipid or fatty acid metabolism. A crucial phase in sample preparation is heating the samples to 100 °C in a Laemmli sample buffer containing SDS before separation by polyacrylamide gel electrophoresis (PAGE). In the current study, the analysis of several proteins was performed following modifications of the heating step during sample preparation. Multiple samples of the human Jurkat cell line were prepared using commercial or homemade Laemmli sample buffer. Samples were subjected to incubation at different temperatures for varying periods of time prior to separation by SDS-PAGE, transfer onto PVDF membranes and detection with specific antibodies. In samples incubated at temperatures of 25 °C, 40 °C, 70 °C and 100 °C, detection of the transmembrane protein elongase of long chain fatty acids 5 (ELOVL5) significantly decreased with temperature to a near total absence of signal at 100 °C. Heating (100 °C) the samples even for 1 min resulted in significant loss of ELOVL5 band intensity that was associated with the appearance of higher molecular weight immunoreactive materials. Loss of ELOVL5 band intensity was also observed with heating of samples at 100 °C prepared from HepG2, HEK293, MCF-7 and SKRB cells. The robust induction of ELOVL5 in stimulated primary T cells was not detected when sample were heated. The detection of fatty acid-metabolizing enzymes stearoyl-CoA desaturase-1 and long-chain-fatty-acid-CoA ligases 3 and 4 showed bands with significantly less intensity after heating at 100 °C compared to samples prepared at room temperature. Heating samples at 100 °C did not affect the detection of transmembrane proteins ERBB2 and five-lipoxygenase activating protein, or the soluble 5-lipoxygenase protein. Overall, the number of transmembrane passes of a protein was not predictive of loss of band intensity after heating, however this study indicates that sample heating can drastically affect the ability to detect proteins following separation by SDS-PAGE. This has implications for any detection methods that follow SDS-PAGE.

Ferroptosis inhibitory constituents from the fruits of Cullen corylifolium

In current study, we studied the phytochemicals of Cullen corylifolium (fruits) in which we identify twenty compounds, including two coumarins (1 and 2), three coumestans (3–5), fourchalcone (6–9), three dihydroflavones (10–12), four isoflavones (13–16), one flavonoid (17) and three meroterpenes (18–20). Among these, compounds 4, 5 and 12 were isolated from C. corylifolium for the first time. The ferroptosis inhibitory effects of the isolated phytochemicals were assessed using erastin-exposed HT22 mouse hippocampal cells. Compounds 3 and 18 showed the most potent inhibition with the IC50 values of 5.21 μM and 5.41 μM, respectively. Moreover, molecular docking study showed that compound 3 possessed tremendous inhibitory affinity for human 5-lipoxygenase (5-LOX) and Kelch-like ECH-related protein 1: nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) protein-protein interactions, two important ferroptosis-related targets. These findings indicate that compound 3 (psoralidin) may be a potential therapeutic agent for the treatment of ferroptosis-related diseases.

Human cytomegalovirus infection is correlated with enhanced cyclooxygenase-2 and 5-lipoxygenase protein expression in breast cancer

PurposeWhile enhanced expression of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO) and their derived metabolites is associated with breast cancer (BC) risk, the precise link between BC carcinogenesis and enhanced inflammatory activity remains to be clarified. Human Cytomegalovirus (HCMV) may induce expression of COX-2 and 5-LO and is frequently found in breast cancer biopsies. Thus, we investigated whether there is an association between HCMV proteins and expression of COX-2 and 5-LO in human BC tissue and BC cell lines.Materials and methodsParaffin embedded biopsies obtained from 49 patients with breast cancer and 26 tissue samples from adjacent, benign breast tissues were retrospectively examined for HCMV-immediate early (IE), HCMV-Late (LA), COX-2, and 5-LO proteins by immunohistochemistry. In vitro, uninfected and HCMV-infected BC cell lines were examined for COX-2 and 5-LO transcripts and proteins by PCR and flow cytometry.ResultsExtensive expression of COX-2, 5-LO and HCMV-IE proteins were preferentially detected in BC samples. We found a statistically significant concordant correlation between extensive HCMV-IE and COX-2 (P < 0.0001) as well as with HCMV-IE and 5-LO (P = 0.0003) in infiltrating BC. In vitro, HCMV infection induced COX-2 and 5-LO transcripts and COX-2 proteins in MCF-7 cells (P =0.008, P =0.018, respectively). In MDA-MB-231 cells that already had high base line levels of COX-2 expression, HCMV induced both COX-2 and 5-LO proteins but not transcripts.ConclusionOur findings demonstrate a significant correlation between extensive HCMV-IE protein expression and overexpression of COX-2 and 5-LO in human breast cancer.

Computational modeling and study of the anti-cancer activity of novel NSAID 1-acyl-4-cycloalkyl/arylsemicarbazide and 1-acyl-5-benzyloxy/hydroxy carbamoylcarbazide derivatives using molecular docking and molecular dynamics simulations

In this study, a series of NSAID 1-acyl-4-cycloalkyl/arylsemicarbazides and 1-acyl-5-benzyloxy/hydroxy carbamoylcarbazides possess anti-cancer activity against three human cancer cell lines L1210, CEM, and HeLa have been investigated using a combined approach including quantitative structure activity relationship (QSAR) study, molecular docking, and molecular dynamics (MD) simulations. First, different molecular descriptors were calculated for these compounds. Stepwise multiple linear regression (MLR) method was performed for selecting some common descriptors, including VEA1, hydration energy (HE), log P, and binding energy for all three cell lines. Then, other QSAR models were constructed using support vector regression (SVR) method. According to the results, SVR models were more efficient in predicting the anti-cancer activity. To better understand the mechanism of the binding interactions of NSAID 1-acyl-4-cycloalkyl/arylsemicarbazide and 1-acyl-5-benzyloxy/hydroxy carbamoylcarbazide derivatives with 5-lipoxygenase (5-LOX) protein, molecular docking studies were conducted. These studies have also been used to explore the effects of HE, log P, and binding energy on anti-cancer activity of the studied compounds. The results of molecular docking suggest that hydrophobic interactions of ligands with the active site of 5-LOX are responsible for their anti-cancer activities. Finally, molecular dynamics (MD) simulations using GROMACS package were used for evaluating the stability of 5-LOX in complex with NSAID 1-acyl-4-cycloalkyl/arylsemicarbazide and 1-acyl-5-benzyloxy/hydroxy carbamoylcarbazide derivatives. The results of MD simulations demonstrate stability of protein structure in complex with active compounds, 12 and 27.

Dormant 5-lipoxygenase in inflammatory macrophages is triggered by exogenous arachidonic acid

The differentiation of resident tissue macrophages from embryonic precursors and that of inflammatory macrophages from bone marrow cells leads to macrophage heterogeneity. Further plasticity is displayed through their ability to be polarized as subtypes M1 and M2 in a cell culture microenvironment. However, the detailed regulation of eicosanoid production and its involvement in macrophage biology remains unclear. Using a lipidomics approach, we demonstrated that eicosanoid production profiles between bone marrow-derived (BMDM) and peritoneal macrophages differed drastically. In polarized BMDMs, M1 and M2 phenotypes were distinguished by thromboxane B2, prostaglandin (PG) E2, and PGD2 production, in addition to lysophospholipid acyltransferase activity. Although Alox5 expression and the presence of 5-lipoxygenase (5-LO) protein in BMDMs was observed, the absence of leukotrienes production reflected an impairment in 5-LO activity, which could be triggered by addition of exogenous arachidonic acid (AA). The BMDM 5-LO regulatory mechanism was not responsive to PGE2/cAMP pathway modulation; however, treatment to reduce glutathione peroxidase activity increased 5-LO metabolite production after AA stimulation. Understanding the relationship between the eicosanoids pathway and macrophage biology may offer novel strategies for macrophage-associated disease therapy.

Targeted deep resequencing of ALOX5 and ALOX5AP in patients with diabetes and association of rare variants with leukotriene pathways.

The aim of the present study was to investigate a possible association between the accumulation of rare coding variants in the genes for arachidonate 5-lipoxygenase (ALOX5) and ALOX5-activating protein (ALOX5AP), and corresponding production of leukotrienes (LTs) in patients with type 2 diabetes mellitus (T2DM) receiving acetylsalicylic therapy. Twenty exons and corresponding introns of the selected genes were resequenced in 303 DNA samples from patients with T2DM using pooled polymerase chain reaction amplification and next-generation sequencing, using an Illumina HiSeq 2000 sequencing system. The observed non-synonymous variants were further confirmed by individual genotyping of DNA samples comprising of all individuals from the original discovery pools. The association between the investigated phenotypes was based on LTB4 and LTE4 concentrations, and the accumulation of rare missense variants (genetic burden) in investigated genes was evaluated using statistical collapsing tests. A total of 10 exonic variants were identified for each resequenced gene, including 5 missense and 5 synonymous variants. The rare missense variants did not exhibit statistically significant differences in the accumulation pattern between the patients with low and high LTs concentrations. As the present study only included patients with T2DM, it is unclear whether the absence of observed association between the accumulation of rare missense variants in investigated genes and LT production is associated with diabetic populations only or may also be applied to other populations.

The 5-Lipoxygenase as modulator of Alzheimer's γ-secretase and therapeutic target.

Alzheimer's disease (AD) is an age-related, neurodegenerative disorder characterized by cognitive impairment with memory loss, extracellular amyloid beta (Aβ) peptides aggregation, and intracellular hyper-phosphorylated tau neurofibrillary tangles (NFT) accumulation. Although the 5-lipoxygenase (5LO) protein enzyme is well known as an important modulators of oxidation and inflammation, recent work has highlighted the new hypothesis that this pathway may play a direct role in AD pathogenesis. In this review article, we will discuss how the 5LO via the γ-secretase influences Aβ peptides formation, and other molecular pathologies including neuroinflammation, synaptic integrity, and cognitive functions, and provide an assessment of how targeting this protein could lead to novel therapeutics for AD and other related neurodegenerative disorders.

The 5-lipoxygenase pathway: oxidative and inflammatory contributions to the Alzheimer's disease phenotype.

Alzheimer’s disease (AD) is the most common, and, arguably, one of the most-well studied, neurodegenerative conditions. Several decades of investigation have revealed that amyloid-β and tau proteins are critical pathological players in this condition. Genetic analyses have revealed specific mutations in the cellular machinery that produces amyloid-β, but these mutations are found in only a small fraction of patients with the early-onset variant of AD. In addition to development of amyloid-β and tau pathology, oxidative damage and inflammation are consistently found in the brains of these patients. The 5-lipoxygenase protein enzyme (5LO) and its downstream leukotriene metabolites have long been known to be important modulators of oxidation and inflammation in other disease states. Recent in vivo evidence using murine knock-out models has implicated the 5LO pathway, which also requires the 5LO activating protein (FLAP), in the molecular pathology of AD, including the metabolism of amyloid-β and tau. In this manuscript, we will provide an overview of 5LO and FLAP, discussing their involvement in biochemical pathways relevant to AD pathogenesis. We will also discuss how the 5LO pathway contributes to the molecular and behavioral insults seen in AD and provide an assessment of how targeting these proteins could lead to therapeutics relevant not only for AD, but also other related neurodegenerative conditions.

Leukotriene production profiles and actions in the bovine endometrium during the oestrous cycle.

We have previously shown the influence of leukotrienes (LTs) on reproductive functions in vivo: LTB4 is luteotrophic and supports corpus luteum function inducing PGE2 and progesterone (P4) secretion, whereas LTC4 is luteolytic and stimulates PGF2α secretion in cattle. The aim of this study was to examine expression and production profiles of LTs and their actions in the endometrium. LT receptors (LTB4R for LTB4 and CysLTR2 for LTC4), 5-lipoxygenase (LO), 12-LO synthase (LTCS) and LTA4 hydrolase (LTAH) mRNA and protein expression, as well as LT production were measured in bovine endometrial tissue during the luteal phases of the oestrous cycle. The action of LTs on uterine function was studied by measuring the level of PGs after stimulating uterine slices with LTs on Days 8-10 of the cycle. Expression of 5-LO and LTB4R mRNA and protein were highest on Days 2-4 of the cycle, while CysLTR2 and LTCS were highest on Days 16-18 (P<0.05). LTB4 concentration was highest on Days 2-4 of the cycle, whereas the greatest LTC4 level was on Days 16-18 (P<0.05). Both LTB4 and C4 increased the content of PGE2 and F2α in endometrial slices at a dose of 10(-7)M (P<0.05). In summary, mRNA expression and activation of receptors for LTB4 and production occur in the first part of the cycle, whereas LTC4 and its receptors predominate at the end of the cycle. The 12-LO and 5-LO pathways are complementary routes of LT production in the bovine uterus.

Neuroinflammation and Alzheimer’s disease: lessons learned from 5-lipoxygenase

AbstractAside from the well-known amyloid beta and tau pathologies found in Alzheimer’s disease (AD), neuroinflammation is a well-established aspect described in humans and animal models of the disease. Inflammatory perturbations are evident not only in neurons, but also in non-neuronal cells and cytokines in the AD brain. Although the amyloid hypothesis implicates amyloid beta (Aβ) as the prime initiator of the AD, brain inflammation in AD has a complex relationship between Aβ and tau. Using our work with the 5-lipoxygenase protein as an example, we suggest that at least in the case of AD, there is an interdependent and not necessarily hierarchical pathological relationship between Aβ, tau and inflammation.

Multiple Antioxidants Improve Cardiac Complications and Inhibit Cardiac Cell Death in Streptozotocin-Induced Diabetic Rats

Diabetic cardiomyopathy, a disorder of the heart muscle in diabetic patients, is one of the major causes of heart failure. Since diabetic cardiomyopathy is now known to have a high prevalence in the asymptomatic diabetic patient, prevention at the earliest stage of development by existing molecules would be appropriate in order to prevent the progression of heart failure. In this study, we investigated the protective role of multiple antioxidants (MA), on cardiac dysfunction and cardiac cell apoptosis in streptozotocin (STZ)-induced diabetic rat. Diabetic cardiomyopathy in STZ-treated animals was characterized by declined systolic, diastolic myocardial performance, oxidative stress and apoptosis in cardiac cells. Diabetic rats on supplementation with MA showed decreased oxidative stress evaluated by the content of reduced levels of lipid per-oxidation and decreased activity of catalase with down-regulation of heme-oxygenase-1 mRNA. Supplementation with MA also resulted in a normalized lipid profile and decreased levels of pro-inflammatory transcription factor NF-kappaB as well as cytokines such as TNF-α, IFN-γ, TGF-β, and IL-10. MA was found to decrease the expression of ROS-generating enzymes like xanthine oxidase, monoamine oxidase-A along with 5-Lipoxygenase mRNA and/or protein expression. Further, left ventricular function, measured by a microtip pressure transducer, was re-established as evidenced by increase in ±dp/dtmax, heart rate, decreased blood pressure, systolic and diastolic pressure as well as decrease in the TUNEL positive cardiac cells with increased Bcl-2/Bax ratio. In addition, MA supplementation decreased cell death and activation of NF-kappaB in cardiac H9c2 cells. Based on our results, we conclude that MA supplementation significantly attenuated cardiac dysfunction in diabetic rats; hence MA supplementation may have important clinical implications in terms of prevention and management of diabetic cardiomyopathy.

Гипоксическое прекондиционирование предупреждает индукцию и активацию 5-липоксигеназы при ишемии и реперфузии сердца крыс

Male Wistar rats were subjected to hypoxic preconditioning (10% O2 in nitrogen for 3 h). In 24 h heart were isolated and subjected to 30 min ischemia and 40 min reperfusion. Changes in expression of 5-lipoxygenase (5-LO) protein in rat heart ventricles, and in myocardial subcellular fractions were evaluated by Western blotting. It was found that hypoxic preconditioning attenuated reperfusion damage of cardiomyocytes with reducing the release of LDH by 27.6%. After ischemia and reperfusion, expression of 5-LO was 10.5-fold elevated in the left ventricle and 14.3-fold - in the right one. During ischemia and reperfusion occurred gradual translocation of 5-LO protein in nuclear subcellular compartment, more expressive in the left ventricle. Hypoxic preconditioning did not significant increase in 5-LO expression, but fully prevented its growth in the following ischemia-reperfusion, and partly reduced protein translocation at reperfusion in the left ventricle. Thus, hypoxic preconditioning limits proinflammatory effects ofischemia and reperfusion in myocardium, preventing the increase in expression of 5-LO, and reducing the alteration of cardiomyocytes.

Association of ALOX5AP with ischemic stroke in eastern Chinese.

5-lipoxygenase protein (ALOX5AP) has been recognized as a susceptibility gene for stroke and coronary artery diseases. The present study was to explore the role of this gene in the eastern Chinese patients with ischemic stroke. Using a case-control design, we studied 658 patients with ischemic stroke and 704 unrelated population-based controls who were age- and sex-matched. The 658 patients were classified by the Trial of Org 10172 in Acute Stroke Treatment (TOAST). Two single-nucleotide polymorphisms (SNPs) covering ALOX5AP were genotyped. The genotype frequencies of TG of the SNPs rs17222919 located in the promoter of the ALOX5AP gene were significantly higher in patients with ischemic stroke than in controls (OR (*)=1.34, 95%CI(*)=1.02-1.75), especially in patients with ischemic stroke caused by small-artery occlusion (SAO) (OR (*)=1.40, 95%CI(*)=1.02-1.93). Meanwhile, the genotype frequencies of TG and TG/GG were higher in female patients than in the controls. After specification, the genotype frequencies of TG and TG/GG were higher in the patients than in controls with hypertension. The genotype frequencies of AG and AG/GG of the SNPs rs9579646 located in the intron of the ALOX5AP gene were higher in the controls than in the patients. After specification, the genotype frequencies of TG were higher in the controls than patients without hypertension. The present study suggests that sequence variants in the ALOX5AP gene are significantly associated with ischemic stroke.

Two dimensional difference gel electrophoresis analysis of cerebrospinal fluid in tuberculous meningitis patients

Tuberculous meningitis (TBM) is a serious complication of tuberculosis that affects the central nervous system. Present methods to diagnose TBM are not suitable for early diagnosis. Molecular markers and sensitive methods to identify them in the early stage of infection of TBM are critically needed for efficient management. We have done the proteomic analysis of TBM cerebrospinal fluid (n=20) with 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 11 human proteins and 8 mycobacterial proteins with changed expression levels in comparison to controls. Arachidonate 5-lipoxygenase and glial fibrillary acidic protein, two of the identified proteins, were validated with western blot technique on a larger set of disease and control samples (n=40). These two proteins were also analyzed in fungal meningitis samples. We suggest that arachidonate 5-lipoxygenase can be considered for validation as a potential marker for diagnosis of TBM.

Expression of 5-1ipoxygenase in colon mucosa of ulcerative colitis

Objective To investigate the expressions of 5-lipoxygenase （5-LOX） protein and mRNA in colon mucosa of ulcerative colitis （UC） and to analyze its correlation with endoscopic and histological grading. Methods The biopsies from 32 UC patients and 26 healthy controls were collected, and the expressions of 5-LOX mRNA and protein in colonic mucosa was determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry assay, respectively. The endoscopic and histological grading of 32 UC patients were also recorded. Results According to endoscopic grading, there were 10 cases of grade Ⅰ , 19 of grade Ⅱ and 3 of grade Ⅲ in 32 UC patients, and there were 19 cases of grade Ⅰ , 9 of grade Ⅱ and 4 of grade Ⅲ according to histological grading. The expressions of 5-LOX mRNA and protein in UC patients were significantly higher than those in healthy controls （P 〈0. 05 ）. The rate of 5-LOX protein expression increased with endoscopic and histological graObjective To investigate the expressions of 5-lipoxygenase （5-LOX） protein and mRNA in colon mucosa of ulcerative colitis （UC） and to analyze its correlation with endoscopic and histological grading. Methods The biopsies from 32 UC patients and 26 healthy controls were collected, and the expressions of 5-LOX mRNA and protein in colonic mucosa was determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry assay, respectively. The endoscopic and histological grading of 32 UC patients were also recorded. Results According to endoscopic grading, there were 10 cases of grade Ⅰ , 19 of grade Ⅱ and 3 of grade Ⅲ in 32 UC patients, and there were 19 cases of grade Ⅰ , 9 of grade Ⅱ and 4 of grade Ⅲ according to histological grading. The expressions of 5-LOX mRNA and protein in UC patients were significantly higher than those in healthy controls （P 〈0. 05 ）. The rate of 5-LOX protein expression increased with endoscopic and histological grades, which was also positively correlated with endoscopic grades （ P 〈 0. 05 ）, but not with histological grades （ P 〉0. 05 ）. Conclusion The expressions of 5-LOX mRNA and protein in colon mucosa from UC patients might be a marker for disease activity. 5-LOX may play a pivotal role in inflammation of UC and be a target site for treatment. Key words: Colitis, ulcerative; 5-lipoxygenase; Reverse transcription polymerase chain reaction; Immunohistochemistry

Hyperoxia Exposure Alters Hepatic Eicosanoid Metabolism in Newborn Mice

Prematurely born infants are often treated with supraphysiologic amounts of oxygen, which is associated with lung injury and the development of diseases such as bronchopulmonary dysplasia. Complimentary responses between the lung and liver during the course of hyperoxic lung injury have been studied in adult animals, but little is known about this relationship in neonates. These studies tested the hypothesis that oxidant stress occurs in the livers of newborn mice in response to continuous hyperoxia exposure. Greater levels of glutathione disulfide and nitrotyrosine were detected in lung tissues but not liver tissues from newborn mice exposed to hyperoxia than in room air-exposed controls. However, early increases in 5-lipoxygenase and cyclooxygenases-2 protein levels and increases in total hydroxyeicosatetraenoic acid and prostaglandin levels were observed in the liver tissues of hyperoxia-exposed pups. These studies indicate that free radical oxidation occurs in the lungs of newborn pups exposed to hyperoxia, and alterations in lipid metabolism could be a primary response in the liver tissues. The findings of this study identify possible new mechanisms associated with hyperoxic lung injury in a newborn model of bronchopulmonary dysplasia and thus open opportunities for research.

Protein expression of 5-lipoxygenase and activation and cytotoxicity of Benzidine in human bronchial epithelial cells

To investigate the effect of intracellular 5-lipoxygenase on oxidation of benzidine in HBE cells and to provide further evidence that lipoxygenase is an alternative pathway for the oxidation of xenobiotics mediated by cytochrome P450. Enzyme system test: Soybean lipoxygenase (SLO), substrate (benzidine) and other components reacted in the enzyme system, followed by detection of the reaction products by spectrophotometry. In vitro test: After HBE cells were exposed to benzidine, the protein levels of 5-lipoxygenase in HBE cells were assessed by Western-blot, and the DNA damage by the single cell gel electrophoresis. At last, the effect of the specific inhibitor of 5-lipoxygenase (AA861) on 5-lipoxygenase protein expression and DNA damage in HBE cells were detected. SLO could catalyze the co-oxidation of benzidine to generate benzidine diimine in the presence of hydrogen peroxide. Under optimal condition, numax value of the oxidation of benzidine catalyzed by SLO was 1.42 nmol*min(-1) SLO, and the Km value of benzidine was 1.48 mmol/L. EGCG could inhibit the oxidation of benzidine by SLO. Benzidine could induce 5-lipoxygenase protein expression in HBE cells, but AA861 was invalid. Benzidine caused DNA damage in HBE cells, which could be significantly inhibited by AA861. 5-LOX protein expression in HBE cells can be regulated by benzidine, which suggests that the co-oxidation of benzidine by 5-LOX could produce into electrophile that could covalently bind to DNA and induce DNA damage, which could be one of the mechanisms for carcinogenesis of BZD. 5-LOX inhibitor AA861 can inhibit this effect.