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The glutathione peroxidase (GPx) family is a major antioxidant enzyme family that catalyzes the reduction of a variety of hydroperoxides. GPxs are divided into selenium- and nonselenium- containing GPxs. Because of their efficient antioxidant activity, which depends on the presence of the amino acid residue selenocysteine, selenium-containing GPxs have been the subject of many studies. However, the physiological roles of the nonselenium GPxs remain unclear. Here, we report that the deletion of phospholipid hydroperoxide glutathione peroxidase (PHGPx) homologues causes accelerated aging that leads to a shortened lifespan in Caenorhabditis elegans. PHGPx is an antioxidant enzyme that directly reduces the phospholipid hydroperoxides generated in biomembranes. The quadruple phgpx mutant gpx-1; gpx-2; gpx-6; gpx-7 developed normally, reached adulthood and reproduced as well as the wild type. However, a lifespan analysis showed that the quadruple phgpx mutant had a short maximum lifespan, with an age-related increase in its mortality rate. The intestine is the primary tissue expressing gpx-1, gpx-2, gpx-6 and gpx-7 in C. elegans, and the expression of gpx-6 is greatly enhanced under starvation conditions. These results suggest that the C. elegans PHGPx homologues have important functions in the regulation of aging, probably by reducing oxidative damage in the intestine. [ABSTRACT FROM AUTHOR]

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674-683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a CreloxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans. [ABSTRACT FROM AUTHOR]

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is transcribed into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar types, from one gene by alternative transcription using different first exons, Ia and Ib. We investigated the regulatory mechanisms of the expressions of the three types of PHGPx using promoter analysis with luciferase as the reporter gene and electrophoretical mobility shift analysis. Here we report a draft of the positive regulatory region and the core promoter regions of PHGPx in several cell lines. From promoter deletion analysis we identified the three distinct core regions of mitochondrial PHGPx, non-mitochondrial PHGPx and nucleolar PHGPx. The core promoter activity of non-mitochondrial PHGPx was high in L929 cells, but relatively low for mitochondrial and nucleolar PHGPx. We also identified the positive regulatory region of mitochondrial PHGPx by deletion and mutation analysis of 5′-flanking regions of mitochondrial PHGPx. This region could regulate the promoter activity of non-mitochondrial PHGPx; however, up-regulation by this region was normally suppressed by the upstream region in somatic cells. Electrophoretical mobility shift analysis demonstrated that a specific transcription factor complex bound to this region in adult testis, but not in young testis and different sizes of complexes bound to this region between testis and brain. [ABSTRACT FROM PUBLISHER]

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is the only known intracellular antioxidant enzyme that can directly reduce lipid hydroperoxide in membrane. Mitochondrial and non-mitochondrial PHGPx and sperm nuclei GPx are transcribed from one gene by alternative transcription using different first exons Ia and Ib, respectively. To examine the role of PHGPx in development, we generated mice deficient in PHGPx by a targeted disruption of all exons of the PHGPx gene. Heterozygotes are viable, fertile, and appear normal, despite having decreased levels of three types of PHGPx mRNA and protein. Embryos homozygous for PHGPx-null die between 7.5 and 8.5 days post coitum (dpc), probably developing distal apoptosis. We examined the expression of PHGPx in mouse embryos using immunohistochemical analysis with anti-PHGPx mAb. The expression of PHGPx was detected in the embryonic ectoderm and the yolk sac membrane at 7.5 dpc. The results demonstrated that PHGPx is expressed in early gastrulation stage at 7.5 dpc and that the expression of PHGPx was essential for normal mouse development. [Copyright &y& Elsevier]

The esterification of endogenously formed 5-hydroxyeicosatetraenoic acid (5-HETE) to cellular lipids in rat polymorphonuclear leukocytes (PMNL) was studied quantitatively by a fluorometric method using HPLC. Rapid and maximal production of free 5-HETE was observed after a 5-min stimulation of PMNL with A23187. The amount of free 5-HETE then declined rapidly, while that of 5-HETE esterified to phospholipids and triacylglycerol concomitantly increased in a time-dependent manner. Stimulation by A23187 yielded approximately 100 ng/107 cells esterified 5-HETE in 60 min, which corresponded to the decrease in the amount of free 5-HETE from 5 min to 60 min and indicated that free 5-HETE, which was formed endogenously, was metabolized predominantly by esterification to cellular lipids. The esterification profile of exogenous 5-HETE was different from that of endogenous 5-HETE. 5-[3H]HETE, which was added exogenously to the culture medium, was rapidly incorporated into PMNL and almost 80% of the total radioactivity was located in triacylglycerol. A quantitative study revealed chat endogenous 5-HETE was esterified equally to phospholipids and triacylglycerol. Like PMNL, peritoneal macrophages treated with A23187 released significant amounts of 5-HETE. However, less 5-HETE was esterified to cellular lipids than in PMNL. Negligible amounts of 12-HETE produced by activated peritoneal macrophages or activated platelets after a challenge with A23187, were esterified during the entire incubation. Exogenous 5-HETE was rapidly taken up by PMNL, but was incorporated into macrophages much more slowly than into PMNL. No uptake of 12-HETE into macrophages was observed. The rapid uptake of exogenous 5-HETE was strongly inhibited by the suppression of acylation at 5-HETE by triaesin C. These results suggest that esterification might be one of the factors that regulate the rate of incorporation of 5-HETE. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria are major compartments in cells responsible for generating reactive oxygen species, which can cause the development of diabetes, Parkinson''s disease and premature aging. Antioxidant systems in mitochondria are important for the prevention of diseases and reduction in the speed of aging. We investigated whether the reactive oxygen species generated in mitochondria induced the expression of metallothionein as an antioxidant. We compared the expression level of metallothionein mRNA in mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx)-overexpressed (PHGPx-ov) cells with that in control cells. These cells were treated with respiratory inhibitors, including rotenone and 2, 4-dinitrophenol; under these conditions, the PHGPx-ov cells were more resistant to cell death than the control cells. In addition, the intracellular reactive oxygen species level that was induced by these inhibitors was lower in PHGPx-ov cells than in control cells. This indicates that PHGPx degrades the membrane phospholipid hydroperoxide that is formed via the reactive oxygen species generated in mitochondria. The enhanced expression of metallothionein-I and metallothionein-II mRNA in rotenone-treated control cells was significantly decreased in rotenone-treated PHGPx-ov cells, suggesting that the hydrogen peroxide that is formed by superoxide anions generated in mitochondria diffuse into the cytosol and induce metallothionein mRNA expression. Conversely, the expression of manganese-superoxide dismutase (Mn-SOD) mRNA, which is localized in mitochondria, was not correlated with the intracellular reactive oxygen species level that was induced by rotenone treatment. These results suggest that metallothionein expression is sensitively and strictly regulated by the oxidative state that is induced by mitochondrial respiration. [Copyright &y& Elsevier]