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PubMed2006

Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-gamma.

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.

Animals↗

PubMed2006

Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex.

Previous reports have demonstrated that new Cre recombinase specificities can be developed for symmetrically designed lox mutants through directed evolution. The development of Cre variants that allow the recombination of true asymmetric lox mutant sites has not yet been addressed, however. In the present study, we demonstrate that a mixture of two different site-specific Cre recombinase molecules (wt Cre and a mutant Cre) catalyzes efficient recombination between two asymmetric lox sites in vitro, presumably via formation of a functionally active heterotetrameric complex. The results may broaden the application of site-specific recombination in basic and applied research, including the custom-design of recombinases for natural, asymmetric, and lox-related target sequences present in the genome. Future applications may potentially include genomic manipulations, for example, site-specific integrations, deletions or substitutions within precise regions of the genomes of mammalians and other organisms.

Animals↗

PubMed2006

Essential structural profile of a dual functional inhibitor against cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX): molecular docking and 3D-QSAR analyses on DHDMBF analogues.

It is recently proposed that compounds with equal capabilities of inhibiting COX and 5-LOX, both are key enzymes involved in the arachidonic acid (AA) cascade, are expected to be safer non-steroidal anti-inflammatory drugs (NSAIDs). To dig out helpful information in designing dual functional inhibitors against the two enzymes, homology modeling, molecular dynamics (MD) simulations, automated docking, and 3D-QSAR analyses were performed in this study on 21 COX-2/5-LOX dual inhibitors, namely, 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran (DHDMBF) analogues. A 3D-model of 5-LOX was built based on the high-resolution X-ray structure of rabbit reticulocyte 15-lipoxygenase. Molecular docking was then applied to locate the binding orientations and conformations of DHDMBF analogues with COX-2 and 5-LOX, respectively, leading to highly predictive CoMFA models constructed on the basis of the binding conformations with q2 values of 0.782 and 0.634 for COX-2 and 5-LOX, respectively. In addition, CoMFA field distributions were found in good agreement with the structural characteristics of the corresponding binding sites. Both the docking simulations and QSAR analyses suggest that new potent dual inhibitors should share a structural feature with a moderately bulky group at R2 position and a rather negatively charged group around the position of the carbonyl group of DHDMBFs. Therefore, the final 3D-QSAR models and the information of the inhibitor-enzyme interaction should be useful in developing new NSAIDs as anti-inflammation drugs with favorable safety profile.

Amino Acid Sequence↗

PubMed2005

Synthesis and biological evaluation of a new class of acyl derivatives of 3-amino-1-phenyl-4,5-dihydro-1H-pyrazol-5-one as potential dual cyclooxygenase (COX-1 and COX-2) and human lipoxygenase (5-LOX) inhibitors.

A series of acyl derivatives of 3-amino-1-phenyl-4,5-dihydro-1H-pyrazol-5-one as potential human 5-LOX and COX 1 and COX-2 inhibitors structurally related to the 1-phenyl-3-pyrazolidinone (phenidone, 1) have been synthesized and the activity against COX-1, COX-2 and human 5-LOX enzymes has been evaluated. All the derivatives showed poor activity against enzymes. These data, together with our previous studies, indicated that phenidone and related compounds are not suitable as human 5-LOX inhibitors and that pyrazoline nucleus should not be considered a good scaffold for inhibitors of human 5-LOX enzyme, suggesting the necessity to revisit the proposed mechanism of action of phenidone (1) in human models.

Animals↗

PubMed2001

Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) supports cell adhesion to fibronectin.

Lectin-like oxidized lipoprotein receptor-1 (LOX-1) is a specific receptor for atherogenic oxidized low density lipoprotein (OxLDL) which belongs to the scavenger receptor family. In the present report, we show that LOX-1 can also support cell adhesion to fibronectin (FN) in a divalent cation-independent fashion. CHO-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO), but not untransfected CHO-K1 cells, can adhere to FN-coated plates, but not to collagen-coated plates, in the presence of EDTA. BLOX-1-CHO adhesion to FN-coated plates can also be suppressed by scavenger receptor ligands, such as OxLDL, polyinosinic acid (poly I), and dextran sulfate, but not by native LDL, acetylated LDL, polycytidylic acid (poly C), or chondroitin sulfate. Cultured bovine aortic endothelial cells can similarly adhere to FN-coated plates, which was inhibited by OxLDL, poly I, and dextran sulfate in the presence of EDTA. LOX-1 may play an important role in cell adhesion to FN in an integrin-independent manner.

Animals↗

PubMed2003

Identification of functional lox sites in the plastid genome.

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.

3' Untranslated Regions↗

PubMed2003

Induction of LOX-1 and iNOS expressions by ischemia-reperfusion of rat kidney and the opposing effect of L-arginine.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a newly identified endothelial cell surface major receptor for oxidatively modified low-density lipoprotein. Progression of arthrosclerosis in the donor organ after organ transplantation is a major problem. We hypothesized that ischemia-reperfusion induces LOX-1. After 1 h ischemia of bilateral kidneys plus 3, 6, or 12 h reperfusion, we first revealed that LOX-1 mRNA expression was increased in renal cortex and medulla at 6 h after reperfusion, which was decreased by L-arginine supplement. Plasma nitric oxide (NO) end-product nitrite plus nitrate and inducible nitric oxide synthase (NOS) expression were increased after reperfusion of 6 h. However, NOS substrate L-arginine did not augment but markedly decreased plasma NO end product, because L-arginine supplement suppressed inducible NOS expression in kidney. We hypothesized that available L-arginine is depleted by ischemia-reperfusion, leading to inducible NOS induction. Ischemia decreased L-arginine levels in kidney and L-arginine supplement increased NO end products in renal cortex in the earliest phase of reperfusion. These results disclosed for the first time that a deficiency in L-arginine by ischemia reperfusion causes uncoupling of constitutive NOS, which induces inducible NOS and LOX-1, implying why L-arginine is effective for stroke or transplantation in preventing atherosclerotic progress.

Animals↗

PubMed2005

Crystallization and preliminary X-ray diffraction study of L-lactate oxidase (LOX), R181M mutant, from Aerococcus viridans.

L-Lactate oxidase (LOX) from Aerococcus viridans is a member of the alpha-hydroxyacid oxidase flavoenzyme family. An X-ray crystallographic study of a LOX mutant in which Arg181 is replaced by Met was initiated in order to understand the functions of the conserved amino-acid residues around the FMN in the enzyme active site. LOX-R181M crystals belong to the tetragonal space group I422, with unit-cell parameters a = b = 192.632, c = 200.263 A, alpha = beta = gamma = 90 degrees. There are four monomers in the asymmetric unit. Diffraction data were collected under cryogenic conditions to 2.44 A resolution from LOX-R181M crystals at BL41XU, SPring-8.

Binding Sites↗

PubMed2006

The 2.1 A structure of Aerococcus viridans L-lactate oxidase (LOX).

The crystal structure of L-lactate oxidase (LOX) from Aerococcus viridans has been determined at 2.1 A resolution. LOX catalyzes the flavin mononucleotide (FMN) dependent oxidation of lactate to pyruvate and hydrogen peroxide. LOX belongs to the alpha-hydroxy-acid oxidase flavoenzyme family; members of which bind similar substrates and to some extent have conserved catalytic properties and structural motifs. LOX crystallized as two tightly packed tetramers in the asymmetric unit, each having fourfold symmetry. The present structure shows a conserved FMN coordination, but also reveals novel residues involved in substrate binding compared with other family members.

Amino Acid Sequence↗

PubMed2000

Inducible expression of LOX-1, a novel receptor for oxidized LDL, in macrophages and vascular smooth muscle cells.

Macrophages appear to take up oxidized low density lipoprotein (Ox-LDL) by multiple receptor-mediated pathways. This study, therefore, has been performed to determine if LOX-1, a novel receptor for Ox-LDL, which was identified in vascular endothelial cells, is also expressed in macrophages. Expression of LOX-1 can be induced after macrophage-like differentiation in human peripheral blood monocytes as well as THP-1 cells. Furthermore, expression of LOX-1 in macrophages is also upregulated by an inflammatory cytokine TNF-alpha, which was shown to be present in atherosclerotic arterial wall. Expression of this novel receptor LOX-1 may play an important role in Ox-LDL uptake and subsequent foam cell formation in macrophages.

Animals↗

PubMed2005

Eplerenone shows renoprotective effect by reducing LOX-1-mediated adhesion molecule, PKCepsilon-MAPK-p90RSK, and Rho-kinase pathway.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) may play an important role in atherosclerosis by inducing leukocyte adhesion molecules, such as intercellular and vascular cell adhesion molecule-1 (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]). We hypothesized that eplerenone, a novel selective aldosterone blocker, produces inhibition of LOX-1-mediated adhesion molecules, suppresses mitogen-activated protein (MAP) kinase and its downstream effector p90 ribosomal S6 kinase (p90RSK) through the protein kinase Cepsilon (PKCepsilon) pathway, and improves endothelial function by inhibition of Rho-kinase in the renal cortex of Dahl salt-sensitive hypertensive (DS) and salt-resistant (DR) rats. Eplerenone (10, 30, and 100 mg/kg per day) was given from the age of 6 weeks to the left ventricular hypertrophy stage (11 weeks) for 5 weeks. At 11 weeks, expression levels of LOX-1, ICAM-1, VCAM-1, and Rho-kinase were higher in DS rats than in DR rats and were decreased by eplerenone. Similarly, upregulated phosphorylation of PKCepsilon, MAP kinase, and p90RSK in DS rats was also inhibited by eplerenone. In contrast, downregulated endothelial nitric oxide synthase mRNA was increased by eplerenone to a similar degree as after treatment with Y-27632, a selective Rho-kinase inhibitor. Eplerenone administration resulted in significant improvement in glomerulosclerosis (eplerenone 10 mg, -61%; 30 mg, -78%; and 100 mg, -84% versus DS; P<0.01, respectively) and urinary protein (10 mg, -78%; 30 mg, -87%; and 100 mg, -88% versus DS; P<0.01, respectively). These results suggest that the renoprotective effects of eplerenone may be partly caused by inhibition of LOX-1-mediated adhesion molecules and PKCepsilon-MAP kinase-p90RSK pathway, and improvement in endothelial function.

Animals↗

PubMed2005

Protective effects of nimesulide (COX Inhibitor), AKBA (5-LOX Inhibitor), and their combination in aging-associated abnormalities in mice.

Several inflammatory processes play a critical role in brain aging and are associated with increased vulnerability to neurodegeneration. Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), two enzymes involved in the oxygenation of the arachidonic acid, are upregulated in the central nervous system during aging and are associated with different aging-related brain pathologies. The present experiment was performed to study the effects of 5-LOX inhibitor, acetyl-11-keto-beta-boswellic acid (AKBA), nimesulide (preferential COX-2 inhibitor), and their combination on cognitive performance of young and aged mice, using elevated plus maze test. Chronic administration of AKBA (100 mg/kg, p.o.) and nimesulide (2.42 mg/kg, p.o.) for 15 days significantly reversed the aging-induced retention deficit in mice. Coadministration of AKBA and nimesulide enhanced the cognitive performance in aged mice when compared with that in per se treatment. The aging-related increase in oxidative damage (increased LPO and decreased GSH) was reversed by AKBA, nimesulide, and their combination. Further, per se COX and LOX inhibitors and their combination did not produce any alteration in gastrointestinal parameters; they also reversed the aging-induced motor dysfunction in the aged animals. On the basis of these observations, present findings indicated that the combination of COX and LOX inhibitors (dual inhibitors) may provide a new therapeutic innovation for the treatment of aging-related brain disorders such as Alzheimer's disease and different motor dysfunctions with adequate gastrointestinal tolerability.

Aging↗

PubMed2005

Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in pseudomonas aeruginosa.

The complete genome of the bacterial pathogen Pseudomonas aeruginosa has now been sequenced, allowing gene deletion, one of the most frequently used methods in gene function study, to be fully exploited. In this study, we combine the sacB-based negative selection system with a cre-lox antibiotic marker recycling method. This methodology allows allelic exchange between a target gene and a gentamicin cassette flanked by the two lox sequences. A tetracycline plasmid expressing the cre recombinase is then introduced in the mutant strain to catalyze the excision of the lox-flanked resistance marker. We demonstrate here the efficiency of the combination of these two methods in P. aeruginosa by successively deleting ExoS and ExoT, which are two genetically independent toxins of the type-three secretion system (TTSS). This functional cre-lox recycling antibiotic marker system can create P. aeruginosa strains with multiple mutations without modifying the antibiotic resistance profile when compared to the parental strain.

Base Sequence↗

PubMed1992

Site-dependent differences in sensitivity of LOX human melanoma tumors in nude rats to dacarbazine and mitozolomide, but not to doxorubicin and cisplatin.

Three model systems involving LOX human malignant melanoma cells in nude rats were used to compare the chemosensitivity of tumors growing in different tissues. Groups of 4-18 rats with either s.c. xenografts, lung tumor colonies, or bone metastases were treated with cisplatin, doxorubicin, dacarbazine, or mitozolomide. The antitumor effect in the s.c. model was expressed as specific growth delay, and in the experimental metastasis studies as relative increase in life span (RILS), calculated on the basis of observed disease-free survival. Cisplatin had a moderate but significant effect on the progression of LOX tumor growth in all three systems. Doxorubicin was clearly more efficacious, but for both drugs tumor-free survivors were rare or absent. Importantly, for each of the compounds the levels of response were roughly the same in all three models, with specific growth delay and RILS values in the range of 0.2-0.3 for cisplatin and 0.5-0.9 for doxorubicin. In contrast, a significant site-dependent difference in sensitivity of the LOX tumors was observed for two alkylating agents. Thus, dacarbazine, which temporarily caused complete regression of s.c. xenografts (specific growth delay = 21.0), showed a moderate activity in the lung tumor model (RILS = 1.0) but had only a limited effect (RILS = 0.4) on bone metastases. Mitozolomide gave a curative effect in 6 of 10 animals with s.c. and in 4 of 4 animals with lung tumors, whereas in the bone metastasis model it was only slightly superior to doxorubicin (RILS = 1.1). In preliminary attempts to elucidate the underlying mechanisms, no site-dependent differences in drug distribution and in two cellular detoxifying systems were detected. The data demonstrate the usefulness of the LOX models for studying the clinically relevant problem of site-dependent tumor response to chemotherapy.

Animals↗

PubMed2004

Downregulation of tissue factor by RNA interference in human melanoma LOX-L cells reduces pulmonary metastasis in nude mice.

Tissue factor (TF) is the membrane receptor of the serine protease coagulation factor VIIa (FVIIa). Formation of the TF/FVIIa complex initiates the coagulation cascade. We used short hairpin RNA (shRNA)-mediated RNA interference to knock down TF expression in the human metastatic melanoma cell line LOX-L. After transfection with the shRNA construct, 3 stable clones with significantly downregulated TF expression were established. They exhibited decreased proliferation in vitro as determined by (14)C thymidine incorporation and soft agar assay. The in vivo metastatic potential was assessed in an experimental pulmonary metastasis model in which cells from different clones were injected into the tail vein of nude mice. The incidence of pulmonary tumors was significantly lower in mice receiving shRNA-expressing cells (33% +/- 15%) than in control mice injected with wild-type cells or cells stably transfected with empty expression vector (90% +/- 10%). The mice injected with TF-downregulated cells had markedly longer survival time (69 +/- 17 days) compared to the control mice (35.6 +/- 5 days; p = 0.03). Thus, reduction of TF levels in LOX-L cells significantly delayed and reduced lung tumor formation. As a first step in elucidating the molecular basis for this effect, we compared the global gene expression profile in TF-downregulated cells and control cells by using cDNA microarray analysis. Forty-four known human genes were found to be significantly upregulated (> 2-fold; p < 0.05) and 228 genes significantly downregulated (>or= 3-fold; p < 0.05) in TF-downregulated cells compared to control cells. The differentially expressed genes encode proteins functioning in transcription, translation, cell communication and cell growth/death. The results provide a basis for investigating molecular mechanisms underlying the effects of TF on the metastatic capacity of LOX-L melanoma cells.

Animals↗

PubMed2002

Suppression of 15-lipoxygenase synthesis by hnRNP E1 is dependent on repetitive nature of LOX mRNA 3'-UTR control element DICE.

Cytidine-rich 15-lipoxygenase differentiation control element (15-LOX DICE) is a multifunctional cis-element found in the 3'-UTR of numerous eukaryotic mRNAs. It binds KH domain proteins of the type hnRNP E and K, thus mediating mRNA stabilization and translational control. Translational silencing is caused by formation of a simple binary complex between DICE and recombinant hnRNP E1 (E1). Electromobility shift assays and sucrose gradient centrifugation demonstrate that rabbit 15-LOX DICE, which is composed of ten subunits of the sequence (CCCCPuCCCUCUUCCCCAAG)10=10R, is able to bind up to ten molecules of E1. Protein/RNA interaction was studied with different subunits and submotifs of the 10R structure. Binding appears to be dependent on the degree of polymerization of the C-clusters (1R<2R<4R<10R), but not on their order. The minimal motif, which still functioned in E1 binding, contained two C-clusters (CCCCPuCCCUCUU). For efficient translational control, E1 binding is a necessary, but not sufficient, condition. Translational inhibition by E1 is only observed when at least a dimeric 2R configuration of the DICE is present in the 3'-UTR of a reporter mRNA. We conclude that binding of at least two E1 molecules activate or expose a binding site to enable the complex to interact with the 5'-end of the mRNA and the translational machinery. DICE-motifs are widely distributed in nature. The UTR database UTRnr contains 78 entries of mRNAs with 15-LOX DICEs. Most DICEs were two- to fourfold repetitive, but also highly repetitive structures were found, as in quail myelin protein mRNA (31 repeats) and hyperglycemic hormone mRNA of two crayfish species (nine and 11 repeats).

3' Untranslated Regions↗

PubMed2003

Association of positional and functional candidate genes FGF1, FBN2, and LOX on 5q31 with intracranial aneurysm.

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22-31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene ( LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (chi(2)=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (chi(2)=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.

Aged↗

PubMed2005

Critical role of bradykinin-eNOS and oxidative stress-LOX-1 pathway in cardiovascular remodeling under chronic angiotensin-converting enzyme inhibition.

To elucidate the molecular mechanisms of the cardioprotective effect of angiotensin-converting enzyme (ACE) inhibitors, we evaluated whether the effect of quinapril involved in bradykinin-endothelial nitric oxide synthase (eNOS) and oxidative stress-lectin-like oxidized LDL receptor-1 (LOX-1) pathway. Dahl salt-sensitive hypertensive (DS) rats were fed a diet containing 8% NaCl and treated with one of the following drug combinations for 5 weeks, from 6 weeks of age to left ventricular hypertrophy stage (11 weeks): vehicle; quinapril; quinapril plus the bradykinin B2 receptor antagonist FR172357; the NAD(P)H oxidase inhibitor apocynin; or quinapril plus apocynin. eNOS expression, which was decreased in hypertrophy stage, was significantly increased by quinapril and/or apocynin, but not by quinapril plus FR172357. Upregulated expression of NAD(P)H oxidase p22phox, p47phox, gp91phox and LOX-1 was significantly decreased by quinapril to a similar degree as after treatment with apocynin, but not by quinapril plus FR172357. Quinapril and/or apocynin treatment effectively ameliorated left ventricular weight and vascular changes such as increase in medial thickness and perivascular fibrosis and suppressed expression of transforming growth factor-beta1, type I collagen and fibronectin mRNA, but not that of quinapril plus FR172357. These results suggest that the ACE inhibitor quinapril may have cardioprotective effects in this model of hypertension mediated at least in part through effects on the bradykinin-eNOS and oxidative stress-LOX-1 pathway.

Acetophenones↗