The upregulation of messenger ribonucleic acids during 17alpha, 20beta-dihydroxy-4-pregnen-3-one-induced ovulation in the perch ovary.
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While progestins appear to be involved in the local ovarian regulation of vertebrate ovulation, their specific role is unclear. In yellow perch (Perca flavescens) the progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20-P), stimulates ovulation in vitro and this induction requires gene activation. Therefore, the perch model was used to isolate progestin-upregulated mRNAs. Perch ovaries were incubated for 32 h with or without 17,20-P (0.1 microg/ml). Messenger ribonucleic acids were isolated from the tissue and used for differential display PCR (DDPCR). From DDPCR, 5 bands were eventually obtained that were verified by Northern analysis to be consistently upregulated by 17,20-P at 32 h. Using these bands, full-length cDNAs were obtained by library screening and completely sequenced. Based on similarity to known sequences, four of the cDNAs presumably encode for perch forms of (1) neprilysin (PNEP-1; 63% identical); (2) a lysyl oxidase-type protein (PLO-2; 43.2% identical); (3) calmodulin (PCAL-1; 100% identical); and (4) a microtubule aggregate-like protein (PMAP-1; 29.6% identical). The fifth cDNA obtained from DDPCR most likely encodes for an egg protein and will be reported separately. Each of the cDNAs was used to probe Northern blots of ovarian mRNA taken at 0, 12, 24, 32 and 42 h of incubation with 17,20-P. This temporal Northern analysis verified that all four were upregulated by 32 h. In addition, PNEP and PMAP transcripts began to increase by 12 h, while PCAL and PLO transcripts remained elevated through 42 h. On Northern blots of RNA from other perch tissues, calmodulin was found in all tissues, PLO mRNA was ovarian specific, and PMAP mRNA was also present in the gills and liver. Multiple transcripts were observed for PNEP, but the ovarian form induced by 17,20-P was only found in high abundance in the heart. To our knowledge, this is the first report that specifically characterizes progestin upregulated mRNAs in the vertebrate ovary at ovulation. (+info)
Lysyl oxidase coupled with catalase in egg shell membrane.
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The activity of lysyl oxidase was found in egg shell membrane (ESM) of hens. The activity was determined by measuring the enzymatic conversion of n-butylamine and Nalpha-acetyl-L-lysine to n-butyraldehyde and Nalpha-acetyl-L-allysine, respectively. ESM lysyl oxidase was significantly inhibited by beta-aminopropionitrile, chelating agents, and deoxygenation, consistent with the known properties of lysyl oxidase. Nevertheless, ESM lysyl oxidase was insoluble in urea solution, suggesting that it complexes with ESM. These findings support previous reports indicating the presence of lysine-derived cross-links in ESM and the necessity of lysyl oxidase located in the isthmus of the hen oviduct for the biosynthesis of ESM. Lysyl oxidase secreted around the egg white from the isthmus may initiate the cross-linking reaction of ESM protein, and remain as the constituent of ESM. Moreover, the H(2)O(2) released by lysyl oxidase in ESM was completely decomposed by coexisting catalase activity. ESM lysyl oxidase activity was greatly elevated in the presence of H(2)O(2), probably due to the O(2) produced by catalase. These findings indicate that lysyl oxidase is coupled with catalase in ESM. This coupling enzyme system was considered to be involved in the biosynthesis of ESM and to protect the embryo against H(2)O(2). (+info)
Activation of chick tendon lysyl oxidase in response to dietary copper.
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Lysyl oxidase (EC 1.4.3.13), a cuproenzyme, can account for 10-30% of the copper present in connective tissue. Herein, we assess the extent to which tissue copper concentrations and lysyl oxidase activity are related because the functional activity of lysyl oxidase and the copper content of chick tendon are both related to dietary copper intake. Chicks (1-d old) were fed diets (basal copper concentration, 0.4 microg/g diet) to which copper was added from 0 to 16 microg/g diet. Liver and plasma copper levels tended to normalize in chickens that consumed from 1 to 4 microg copper/g of diet, whereas tendon copper concentrations suggested an unusual accumulation of copper in chickens that consumed 16 microg copper/g diet. The molecular weight of lysyl oxidase was also estimated using matrix-assisted laser desorption ionization/time-of-flight/mass spectrometry (MALDI/TOF/MS). A novel aspect of these measurements was estimation of protein mass directly from the surface of chick tendons and aortae. Whether copper deficiency (0 added copper) or copper supplementation (16 microg copper/g of diet) caused changes in the molecular weight of protein(s) in tendon corresponding to lysyl oxidase was addressed. The average molecular weight of the peak corresponding to lysyl oxidase in tendon and aorta from copper-deficient birds was 28,386 Da +/- 86, whereas the average molecular weight of corresponding protein in tendon from copper-supplemented birds was 28,639 Da +/- 122. We propose that the shift in molecular weight is due in part to copper binding and the formation of lysyl tyrosyl quinone, the cofactor at the active site of lysyl oxidase. (+info)
Regulation of lysyl oxidase by interferon-gamma in rat aortic smooth muscle cells.
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Lysyl oxidase is an essential catalyst for the cross-linking of extracellular collagen and elastin. Abnormalities in lysyl oxidase activity may contribute to the pathogenesis of arterial diseases characterized by abnormal matrix remodeling. This study tested the hypothesis that interferon (IFN)-gamma, a proinflammatory cytokine present in aortic aneurysm and arteriosclerotic plaque rupture, downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells. Steady-state lysyl oxidase mRNA levels decreased in a concentration- and time-dependent manner to 30% of control levels after 24 hours of treatment with IFN-gamma. Cell layer lysyl oxidase activity decreased in parallel with the observed changes in steady-state mRNA. Nuclear runoff studies suggested that transcriptional regulation was responsible for at least 40% of the observed downregulation. mRNA decay studies suggested that IFN-gamma also decreased lysyl oxidase mRNA half-life from 9 to 6 hours. Downregulation of lysyl oxidase by IFN-gamma did not appear to require new protein synthesis. This study documents that IFN-gamma downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells by transcriptional and posttranscriptional mechanisms. If similar regulation occurs in vivo, it is possible that IFN-gamma-mediated changes in lysyl oxidase may contribute to arterial diseases characterized by abnormal extracellular matrix. (+info)
Dexamethasone enhances ras-recision gene expression in cultured murine fetal lungs: role in development.
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We have shown that dexamethasone (Dex) accelerates maturation and differentiation of cultured fetal murine lungs (Cilley RE, Zgleszewski SE, Krummel TM, and Chinoy MR. Surg Forum 47: 692-695, 1996). We now demonstrate that although Dex inhibits thinning of acinar walls and secondary septa formation, it does, however, promote lung growth. CD-1 murine fetal lungs were cultured for 7 days in the presence and absence of 10 nM Dex. Dex-modulated genes were investigated and identified by differential display of mRNAs performed with specific anchor primer H-T(11)G and 24 arbitrary primers. Thirty-five differentially expressed cDNAs were isolated, subcloned, sequenced, and identified through BLAST searches. One of these cDNAs, termed Dex2, with enhanced expression in Dex-treated lungs, had 100% similarity with ras-recision gene (rrg), also known as the lysyl oxidase (LOX) gene that encodes lysyl oxidase. LOX gene is very highly conserved, with significant sequence similarity among mouse, rat, and human. Two other cDNAs, termed Dex1 and Dex4, were also identified as rrg, with 92 and 97% sequence similarity with the existing data bank sequence of rrg. LOX enzyme is known to downregulate p21(ras) protein and play a central role in the maturation of collagen and elastin in the extracellular matrix as well as modulate the cytoskeletal elements. Thus LOX may be important in lung developmental processes involving epithelial-mesenchymal interactions. (+info)
Lysyl oxidase activates the transcription activity of human collagene III promoter. Possible involvement of Ku antigen.
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Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin. Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues. Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene. Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay. The effect was specific, because other promoters were not affected. Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity. Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression. The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments. We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70. This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis. (+info)
Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1.
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Mitogenic growth factors and transforming growth factor beta1 (TGF-beta1) induce the generation of reactive oxygen species (ROS) in nonphagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta1 to activate a cell surface-associated NADH:flavin:O(2) oxidoreductase (NADH oxidase) that generates extracellular H(2)O(2). In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta1 with respect to the primary reactive species produced (O(2)(.-) vs. H(2)O(2)), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O(2)(.-) without detectable NADH oxidase activity or extracellular H(2)O(2) release. In contrast, TGF-beta1 does not stimulate intracellular O(2)(.-) production and the delayed induction of extracellular H(2)O(2) release is not associated with O(2)(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O(2)(.-) generation but has no effect on TGF-beta1-induced NADH oxidase activation/H(2)O(2) production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects depending on the primary reactive species generated and site of production. (+info)
Lysyl oxidase and MMP-2 expression in dehydroepiandrosterone-induced polycystic ovary in rats.
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Polycystic ovary syndrome (PCOS) is characterized by cystogenesis; however, the cause of this cystogenesis is unknown. At ovulation, preovulatory collagenolytic activities in the ovarian follicles increase and various proteinases are needed to degrade the tissues surrounding the follicles. To clarify the roles of enzymes in collagen degradation of the follicular wall of polycystic ovary (PCO) in relation to the cystogenesis, we examined expression of lysyl oxidase (LOX), which initiates cross-link formation of the collagen and elastin in the extracellular matrix, and expression of matrix metalloproteinases (MMPs) in ovaries of model rats with PCO induced by dehydroepiandrosterone (DHEA) compared with MMP expression in control rats. DHEA treatment increased LOX mRNA expression to more than three times the control value (P: < 0.01). MMP-2 mRNA expression in control rats was threefold greater than that in the DHEA-induced group (P: < 0.05). Expression of both latent and active forms of MMP-2 in controls was more than twice that in the DHEA-induced group (P: < 0.05) as shown by Western blotting, and expression of the active form of MMP-2 was also twice as high in the controls as in the DHEA-treated group (P: < 0.05) as shown by zymography. Our results suggest that depression of MMP-2 activity and increased LOX expression may be one of the causes of the cystogenesis of PCO. (+info)