Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: C44C10 .12
 59,182 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

5-Lipoxygenase (EC 1.13.11.34) is the key enzyme in the regulation of leukotriene synthesis. Here, the effects of various substituted nitrobenzene compounds on 5-lipoxygenase activity and the formation of leukotriene B4 (LTB4) were studied in polymorphonuclear leukocytes (PMNL), B lymphocytes, and human whole blood. 1-Chloro-2,4,6-trinitrobenzene (TNCB) was found to inhibit calcium ionophore A23187-induced leukotriene synthesis in PMNL in a biphasic manner. Thus, 1.0 microM TNCB caused 50% inhibition of LTB4 formation, but only 16% inhibition was found at 10 times higher concentration. In contrast, this higher concentration of TNCB activated the synthesis of LTB4 when PMNL were stimulated with arachidonic acid alone, demonstrating that TNCB can exert both stimulatory and inhibitory effects on leukotriene synthesis depending on the experimental conditions. The inhibitory effect of 1.0 microM TNCB on ionophore A23187-induced leukotriene synthesis could be circumvented by addition of exogenous arachidonic acid. At high concentrations of TNCB (25-100 microM), the drug blocked ionophore A23187-induced leukotriene synthesis. TNCB also inhibited LTB4 formation in B lymphocytes, as well as in human whole blood. The activity of recombinant 5-lipoxygenase was inhibited by TNCB, and reduced glutathione or beta-mercaptoethanol counteracted this inhibition. This suggests that TNCB might inhibit 5-lipoxygenase by alkylating thiol groups. TNCB possessed a high specificity for 5-lipoxygenase with only modest inhibitory effects on 12-lipoxygenase (EC 1.13.11.31), 15-lipoxygenase (EC 1.13.11.12), and phospholipase A2 (EC 3.1.1.4) activities. Taken together, these results show that TNCB can both specifically inhibit and stimulate leukotriene formation and might be useful in further studies on the regulation of 5-lipoxygenase.
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PMID:Effects of 1-chloro-2,4,6-trinitrobenzene on 5-lipoxygenase activity and cellular leukotriene synthesis. 958 59

Nicotiana tabacum 46-8 cultivar displays an incompatible interaction with race 0 of Phytophthora parasitica var. nicotianae (Ppn), a fungal pathogen of most tobacco cultivars. At the plant level, incompatibility is characterized by the induction of lipoxygenase (LOX, EC = 1.13.11.12) activity and localized hypersensitive cell death before defense gene activation. To evaluate the involvement of LOX in the onset of plant defense, tobacco 46-8 plants were genetically engineered using full-length or partial-length antisense (AS) tobacco LOX cDNA constructs. AS expression strongly reduced elicitor- and pathogen-induced LOX activity. Eight independent AS-LOX lines were selected and assayed for their response to Ppn. After root or stem inoculation with race 0, all AS-LOX lines but one displayed a compatible phenotype whereas control transformed plants, not containing the AS-LOX cassette, showed the typical incompatible reaction. The presence of the fungus in transgenic lines was demonstrated by PCR amplification of a Ppn-specific genomic sequence. A linear relationship was found between the extent of LOX suppression and the size of the lesion caused by the fungus. The AS-LOX plants also showed enhanced susceptibility toward the compatible fungus Rhizoctonia solani. The results demonstrate the strong involvement of LOX in the establishment of incompatibility in plant-microorganism interactions, consistent with its role in the defense of host plants.
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PMID:The incompatible interaction between Phytophthora parasitica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenase sequences. 960 Oct 5

Inhibitors of 5-lipoxygenase-activating protein (FLAP) have been found to induce apoptosis. The current study examined the expression of FLAP and bcl family proteins and the induction of apoptosis in interleukin-3-dependent control and bcl-xL-overexpressing FL5.12 cell lines after treatment with MK886, a specific FLAP inhibitor. FL5.12 cells contained a substantial amount of FLAP protein and mRNA but surprisingly had no measurable 5-lipoxygenase protein or 5-, 12-, or 15-lipoxygenase activity. The basal level of FLAP protein in cells overexpressing bcl-xL was 70% less than in controls. FLAP disappeared 4 h after withdrawal of interleukin-3 in bcl-xL cells but not in control cells, which underwent apoptosis. A dose- and time-response study revealed that 5 nmol of MK886/10(6) cells was sufficient to induce apoptosis both in control and bcl-xL cells, respectively, but to different degrees. bcl-xL and bcl-2 proteins, but not bax or FLAP, were decreased by 4 h after 5 nmol of MK886/10(6) cells in both cell lines, although the higher levels of bcl-xL in overexpressors took longer to disappear. This early loss of bcl-xL and bcl-2 was not attributable to generalized proteolysis, as shown by Coomassie Blue staining and by the maintenance of bax. Caspase-3 was activated 2 h after MK886 treatment in control cells but not in bcl-xL cells. Inhibition of caspase-3 decreased MK886-induced apoptosis by 50% in control cells. Inhibition of this caspase after MK886 treatment was unable to prevent the loss of bcl-xL in control cells but did provide partial protection for the loss of the transfected form, but not the endogenous form, in overexpressing cells. These data indicate that MK886 induces extensive apoptosis that is partially caspase-3 dependent and may be related to a rapid loss of bcl-xL. Although caspase-3 inhibitors had no effect on the loss of bcl-xL, other caspases or protease systems may still be involved. The absence of 5-lipoxygenase in cells containing FLAP, the lower level of FLAP in bcl-xL cells, the apoptosis-inducing activity of MK886, and the rapid loss of bcl-xL and bcl-2 proteins after treatment with MK886 strongly indicate that FLAP has activities unrelated to lipoxygenase and suggest a possible functional or regulatory link between these proteins, which share similar subcellular localizations.
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PMID:A relationship between 5-lipoxygenase-activating protein and bcl-xL expression in murine pro-B lymphocytic FL5.12 cells. 977 36

From a rabbit reticulocyte library a full length cDNA was isolated which predicted a novel lipoxygenase (LOX) sharing 99% identical amino acids with the rabbit 15-lipoxygenase. HPLC product analysis of the bacterially expressed protein identified it as a leukocyte-type 12-lipoxygenase (1.12-LOX). This proves the co-expression of a 15-lipoxygenase and a 1.12-lipoxygenase in one mammalian species. Among the six amino acids that are different to rabbit 15-lipoxygenase, leucine 353 is shown to be the primary determinant for 12-positional specificity. In the 3'-untranslated region of the 12-LOX-mRNA a CU-rich, 20-fold repetitive element has been found, closely related to the differentiation control element (DICE) of the rabbit 15-LOX-mRNA which is organized by ten repeats of 19 bases. By genomic PCR the 3'-terminal part of the gene for the novel 12-lipoxygenase containing the introns 10-13 has been amplified and sequenced. The introns were very similar in length to the corresponding 15-lipoxygenase introns with 89% to 95% identical nucleotide sequences. By screening a rabbit reticulocyte library an alternative 15-lipoxygenase transcript of 3.6 kb has been detected containing a 1019 nucleotides longer 3'-untranslated region (UTR2) than the main 2.6 kb mRNA. The determination of the tissue distribution by Northern blotting showed that the 3.6 kb mRNA2 was only expressed in non-erythroid tissues, whereas the 2.6 kb mRNA1 was exclusively expressed in reticulocytes. The only cell type which has been found to express the 1.12-lipoxygenase abundantly are monocytes. The results indicate that the expression of 1.12-lipoxygenase and 15-lipoxygenase is highly regulated. The UTR2 of the 15-LOX-mRNA2 contained a novel eight-fold repetitive CU-rich motif of 23 bases length which is related but not identical to the DICE of 19 bases in the UTR1. The analysis of a genomic recombinant of the complete 9.0 kb Alox15 gene confirmed that UTR1 and UTR2 are not interrupted by an additional intron.
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PMID:Expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits. 1008 82

The ability of various inhibitors of lipoxygenase (LOX) enzymes and 5-lipoxygenase-activating protein (FLAP) to induce apoptosis has implicated these pathways in the mechanism(s) of this form of cell death. Although FLAP plays an important role in 5-LOX activity, this protein is found at high levels in some cells lacking LOX, suggesting it might mediate other effects. Furthermore, the concentration of MK886, a FLAP inhibitor, required to induce apoptosis is approximately 100-fold more than that required to inhibit LOX, and this compound remains effective in cells lacking LOX. The present study examines the role of FLAP in MK886-induced apoptosis. MK886 induced apoptosis in WSU cells, a human chronic lymphocytic leukaemia cell line that lacks FLAP protein and mRNA, suggesting that this agent is acting independently of FLAP. This conclusion was further supported by the fact that a more specific FLAP inhibitor, MK591, induced only minimal apoptosis in FL5.12 cells, a murine prolymphoid cell line containing FLAP. The role of FLAP was examined more directly by decreasing its expression by more than 50% in FL5.12 cells treated with 10 microM of an antisense oligonucleotide for 48h. This change in FLAP was not accompanied by any increase in apoptosis. Furthermore, FLAP-depleted cells exhibited the same level of apoptosis 8 h after treatment with 10 microM MK886, as did control cells. The increased fluorescence seen in MK886-treated cells loaded with carboxydichlorofluorescein indicates that oxidative reactions are stimulated by this compound, possibly via the release of fatty acids from fatty acid-binding proteins and their subsequent oxidation.
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PMID:The 5-lipoxygenase-activating protein (FLAP) inhibitor, MK886, induces apoptosis independently of FLAP. 1033 77

Plant lipoxygenases (LOX, EC 1.13.11.12) have been involved in processes such as stress responses and development. The levels of these enzymes and their corresponding mRNAs are modulated during these processes as well as by different effectors such as jasmonic acid (JA), its methyl ester (MeJA) or abscisic acid (ABA). A new lipoxygenase (LOX) cDNA clone, PvLOX2, was isolated from a Phaseolus vulgaris nodule library and used to study the LOX mRNA accumulation pattern in some developmental stages and in plants subjected to hormone and stress treatments. In nodules, LOX mRNA reaches a maximum level around day 14 to 16 after Rhizobium tropici inoculation, as compared with LOX mRNA present in uninoculated and inoculated roots at the same days. LOX antigen is detected in the nodule parenchyma and in the uninfected cells. During germination, bean LOX transcripts start to accumulate 48 h after imbibition, remains at the same level until 72 h after imbibition and then declines. In hypocotyl, LOX mRNA is abundant in the growing region and almost absent in the mature region. After water stress or ABA treatment, this mRNA increases in the mature region and decreases in the growing region. In bean seedlings, LOX mRNA is accumulated in response to some types of stresses such as cold and desiccation. Wounding, MeJA or ABA treatment of mature leaves also induces LOX mRNA accumulation. These results indicate that in common bean plants LOX is required during development and stress conditions.
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PMID:Analysis of lipoxygenase mRNA accumulation in the common bean (Phaseolus vulgaris L.) during development and under stress conditions. 1055 5

Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the molecule. The loss of enzyme activity was much earlier than the structural loss of the molecule during the course of unfolding, with the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively. The equilibrium unfolding transition could be adequately fitted to a three-state, two-step model (N left arrow over right arrow I left arrow over right arrow U) and the intermediate fraction was maximally populated at 6.3 M urea. The free energy change (DeltaG(H(2)O)) for the unfolding of native (N) to intermediate (I) was 14.2 +/- 0.28 kcal/mol and for the intermediate to the unfolded state (U) was 11.9 +/- 0.12 kcal/mol. The ANS binding measurements as a function of urea concentration indicated that the maximum binding of ANS was in 6.3 M urea due to the exposure of hydrophobic groups; this intermediate showed significant amount of tertiary structure and retained nearly 60% of secondary structure. The limited proteolysis measurements showed that the initiation of unfolding was from the C-terminal domain. Thus, the stable intermediate observed could be the C-terminal domain unfolded with exposed hydrophobic domain-domain interface. Limited proteolysis experiments during refolding process suggested that the intermediate refolded prior to completely unfolded LOX1. These results confirmed the role of cysteine residues and domain-domain interactions in the reversible unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group.
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PMID:Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1. 1058 2

Nordihydroguaiaretic acid (NDGA) induces apoptosis in a variety of cell lines. The mechanism(s) of this effect is not known, although the focus has been on the ability of NDGA to inhibit lipoxygenase (LOX) activities. In the present study, NDGA-induced apoptosis was studied in a murine hematopoietic cell line, FL5.12. Although this cell line lacks detectable LOX protein or activities, NDGA (10 microM) was able to induce apoptosis. There was a massive loss of mitochondrial membrane potential by 4 h after the addition of NDGA, suggesting that this organelle might be targeted by NDGA. A pro-oxidant NDGA effect has been suggested as playing a role in apoptosis. This was supported by the findings that glutathione disulfide levels were increased by 4 h following treatment with 10 microM NDGA, that pretreatment with N-acetylcysteine completely blocked the NDGA-induced loss of membrane potential and apoptosis, and that lipid peroxidation was enhanced in cells treated with NDGA. However, no evidence of increased levels of reactive oxygen could be seen in NDGA-treated cells loaded with dichlorofluorescin diacetate or dihydrorhodamine and analyzed by flow cytometry. Bcl-X(L) protein levels were unaffected by NDGA treatment. Caspase-3 was rapidly activated with a peak at 8 h after FL5.12 cells were treated with NDGA. Ac-DEVD-CHO (25 microM) and boc-asp-FMK (20 microM) both inhibited caspase-3 enzyme activity by 97% 8 h after NDGA treatment. Boc-asp-FMK, a more general caspase inhibitor, delayed NDGA-induced apoptosis while Ac-DEVD-CHO, a more specific inhibitor of caspase-3, had no effect. These results suggest that NDGA-induced apoptosis happens through reactions that depolarize mitochondria, oxidize glutathione and lipids, but do not generate significant amounts of free reactive oxygen species.
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PMID:Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells. 1065 24

A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid.
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PMID:Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies. 1080 41

A novel membrane lipoxygenase (LOX; EC 1.13.11.12) from eggplant (Solanum melongena L. cv. Belleza negra) fruit chloroplasts has been purified 20-fold to a specific activity of 207 enzymatic units per mg of protein with a yield of 72%. The purification was carried out by sonicating the chloroplastic membranes in the presence of Triton X-114 followed by phase partitioning and anion exchange chromatography. The purified membrane LOX preparation consisted of a single major band with an apparent molecular mass of 97 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results obtained using intact chloroplasts indicate that the enzyme is not localized in the stroma. When the enzyme reacts with linoleic acid, it produces a single peak, which comigrates with standard 9-hydroperoxy-octadecadienoic acid. A physiological role for this chloroplastic LOX is proposed.
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PMID:Purification of a novel lipoxygenase from eggplant (Solanum melongena) fruit chloroplasts. 1124 Sep 10


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