Toshihiro Kurahashi

Germination of photoblastic lettuce seeds is regulated via the control of endogenous physiologically active gibberellin content, rather than of gibberellin responsiveness

Phytochrome regulates lettuce (Lactuca sativa L. cv. Grand Rapids) seed germination via the control of the endogenous level of bioactive gibberellin (GA). In addition to the previously identified LsGA20ox1, LsGA20ox2, LsGA3ox1, LsGA3ox2, LsGA2ox1, and LsGA2ox2, five cDNAs were isolated from lettuce seeds: LsCPS, LsKS, LsKO1, LsKO2, and LsKAO. Using an Escherichia coli expression system and functional assays, it is shown that LsCPS and LsKS encode ent-copalyl diphosphate synthase and ent-kaurene synthase, respectively. Using a Pichia pastoris system, it was found that LsKO1 and LsKO2 encode ent-kaurene oxidases and LsKAO encodes ent-kaurenoic acid oxidase. A comprehensive expression analysis of GA metabolism genes using the quantitative reverse transcription polymerase chain reaction suggested that transcripts of LsGA3ox1 and LsGA3ox2, both of which encode GA 3-oxidase for GA activation, were primarily expressed in the hypocotyl end of lettuce seeds, were expressed at much lower levels than the other genes tested, and were potently up-regulated by phytochrome. Furthermore, LsDELLA1 and LsDELLA2 cDNAs that encode DELLA proteins, which act as negative regulators in the GA signalling pathway, were isolated from lettuce seeds. The transcript levels of these two genes were little affected by light. Lettuce seeds in which de novo GA biosynthesis was suppressed responded almost identically to exogenously applied GA, irrespective of the light conditions, suggesting that GA responsiveness is not significantly affected by light in lettuce seeds. It is proposed that lettuce seed germination is regulated mainly via the control of the endogenous content of bioactive GA, rather than the control of GA responsiveness.

Oxidative stress triggers lipid droplet accumulation in primary cultured hepatocytes by activating fatty acid synthesis

Despite the impaired intestinal lipid absorption and low level of visceral fat, the Sod1-deficient mouse is susceptible to developing liver steatosis. To gain insights into the mechanism responsible for this abnormal lipid metabolism, we analyzed primary cultured hepatocytes obtained from Sod1-deficient and wild-type mice. Lipid droplets began to accumulate in the cultured hepatocytes and was further increased by a Sod1 deficiency. Levels of enzymes involved in lipogenesis were elevated. It thus appears that lipogenesis is activated by oxidative stress, which is more prominent in the case of Sod1 deficiency, and appears to participate in liver steatosis.

An SOD1 deficiency enhances lipid droplet accumulation in the fasted mouse liver by aborting lipophagy

Under normal feeding conditions, oxidative stress stimulates lipid droplets accumulation in hepatocytes. We found that, despite the low visceral fat in Sod1-knockout (KO) mouse, lipid droplets accumulate in the liver to a greater extent than for the wild-type mouse upon fasting. Liver damage became evident in the KO mice. While fasting caused substantial endoplasmic reticulum stress in KO mice, the expression of genes involved in fatty acid production was suppressed. LC3-II, which is essential for the dynamic process of autophagosome formation, was activated in the wild-type mouse and enhanced in the KO mouse. However, the p62, an adapter protein with the ubiquitin- and LC3-binding activity, accumulated abnormally in the livers of KO mice, implying an abortive lipophagic process as the cause for the impaired lipid metabolism and the hepatic damage that occurs upon fasting.

A malfunction in triglyceride transfer from the intracellular lipid pool to apoB in enterocytes of SOD1-deficient mice

We compared lipid metabolism in the intestines of Sod1‐knockout mice with that found in wild‐type mice to elucidate the impact of oxidative stress in vivo. A high‐fat diet in wild‐type mice induced postprandial hypertriglyceridemia, but this adaptive response was impaired in Sod1‐knockout mice. While fewer triglycerides were secreted to the blood in the form of triglyceride‐rich lipoprotein, more lipid droplets accumulated in the enterocytes of Sod1‐knockout mice fed a high‐fat diet. These data collectively suggest that high‐fat diet induces oxidative stress, inhibits lipid secretion to the blood, and ultimately leads to dysfunctional lipid metabolism in enterocytes.

Physiological and pathological views of peroxiredoxin 4.

Peroxiredoxins (PRDXs) form an enzyme family that exhibits peroxidase activity using electrons from thioredoxin and other donor molecules. As the signaling roles of hydrogen peroxide in response to extracellular stimuli have emerged, the involvement of PRDX in the hydrogen peroxide-mediated signaling has become evident. Among six PRDX members in mammalian cells, PRDX4 uniquely possesses a hydrophobic signal peptide at the amino terminus, and, hence, it undergoes either secretion or retention by the endoplasmic reticulum (ER) lumen. The role of PRDX4 as a sulfoxidase in ER is now attracting much attention regarding the oxidative protein folding of nascent proteins. Contrary to this role in the ER, the functional significance of PRDX4 in the extracellular milieu is virtually unknown despite its implications as a biomarker under pathological conditions in some diseases. Other than its systemically expressed form, a variant form of PRDX4 is transcribed from the upstream promoter/exon 1 of the systemic promoter/exon 1 and is uniquely expressed in sexually matured testes. Circumstantial evidence, together with deduced functions from the systemic form, suggests that there are potential roles for testicular PRDX4 in the reproductive processes such as the regulation of hormonal signals and the oxidative packaging of sperm chromatin. Elucidation of these PRDX4 functions under in vivo situations is expected to show the whole picture of how PRDX4 has evolved in multicellular organisms.

Ascorbic acid prevents acetaminophen-induced hepatotoxicity in mice by ameliorating glutathione recovery and autophagy.

Aldehyde reductase (AKR1A) plays a role in the biosynthesis of ascorbic acid (AsA), and AKR1A-deficient mice produce about 10-15% of the AsA that is produced by wild-type mice. We found that acetaminophen (AAP) hepatotoxicity was aggravated in AKR1A-deficient mice. The pre-administration of AsA in the drinking water markedly ameliorated the AAP hepatotoxicity in the AKR1A-deficient mice. Treatment of the mice with AAP decreased both glutathione and AsA levels in the liver in the early phase after AAP administration, and an AsA deficiency delayed the recovery of the glutathione content in the healing phase. While in cysteine supply systems; a neutral amino acid transporter ASCT1, a cystine transporter xCT, enzymes for the transsulfuration pathway, and autophagy markers, were all elevated in the liver as the result of the AAP treatment, the AsA deficiency suppressed their induction. Thus, AsA appeared to exert a protective effect against AAP hepatotoxicity by ameliorating the supply of cysteine that is available for glutathione synthesis as a whole. Because some drugs produce reactive oxygen species, resulting in the consumption of glutathione during the metabolic process, the intake of sufficient amounts of AsA would be beneficial for protecting against the hepatic damage caused by such drugs.

Oxidative stress as a potential causal factor for autoimmune hemolytic anemia and systemic lupus erythematosus

The kidneys and the blood system mutually exert influence in maintaining homeostasis in the body. Because the kidneys control erythropoiesis by producing erythropoietin and by supporting hematopoiesis, anemia is associated with kidney diseases. Anemia is the most prevalent genetic disorder, and it is caused by a deficiency of glucose 6-phosphate dehydrogenase (G6PD), for which sulfhydryl oxidation due to an insufficient supply of NADPH is a likely direct cause. Elevated reactive oxygen species (ROS) result in the sulfhydryl oxidation and hence are another potential cause for anemia. ROS are elevated in red blood cells (RBCs) under superoxide dismutase (SOD1) deficiency in C57BL/6 mice. SOD1 deficient mice exhibit characteristics similar to autoimmune hemolytic anemia (AIHA) and systemic lupus erythematosus (SLE) at the gerontic stage. An examination of AIHA-prone New Zealand Black (NZB) mice, which have normal SOD1 and G6PD genes, indicated that ROS levels in RBCs are originally high and further elevated during aging. Transgenic overexpression of human SOD1 in erythroid cells effectively suppresses ROS elevation and ameliorates AIHA symptoms such as elevated anti-RBC antibodies and premature death in NZB mice. These results support the hypothesis that names oxidative stress as a risk factor for AIHA and other autoimmune diseases such as SLE. Herein we discuss the association between oxidative stress and SLE pathogenesis based mainly on the genetic and phenotypic characteristics of NZB and New Zealand white mice and provide insight into the mechanism of SLE pathogenesis.

Overexpression of Peroxiredoxin 4 Affects Intestinal Function in a Dietary Mouse Model of Nonalcoholic Fatty Liver Disease

Background Accumulating evidence has shown that methionine- and choline-deficient high fat (MCD+HF) diet induces the development of nonalcoholic fatty liver disease (NAFLD), in which elevated reactive oxygen species play a crucial role. We have reported that peroxiredoxin 4 (PRDX4), a unique secretory member of the PRDX antioxidant family, protects against NAFLD progression. However, the detailed mechanism and potential effects on the intestinal function still remain unclear. Methods & Results Two weeks after feeding mice a MCD+HF diet, the livers of human PRDX4 transgenic (Tg) mice exhibited significant suppression in the development of NAFLD compared with wild-type (WT) mice. The serum thiobarbituric acid reactive substances levels were significantly lower in Tg mice. In contrast, the Tg small intestine with PRDX4 overexpression showed more suppressed shortening of total length and villi height, and more accumulation of lipid in the jejunum, along with lower levels of dihydroethidium binding. The enterocytes exhibited fewer apoptotic but more proliferating cells, and inflammation was reduced in the mucosa. Furthermore, the small intestine of Tg mice had significantly higher expression of cholesterol absorption-regulatory factors, including liver X receptor-α, but lower expression of microsomal triglyceride-transfer protein. Conclusion Our present data provide the first evidence of the beneficial effects of PRDX4 on intestinal function in the reduction of the severity of NAFLD, by ameliorating oxidative stress-induced local and systemic injury. We can suggest that both liver and intestine are spared, to some degree, by the antioxidant properties of PRDX4.

Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction

Abstract In response to sustained damage to a kidney, fibrosis that can be characterized as the deposition of a collagenous matrix occurs and consequently causes chronic kidney failure. Because most animals used in experiments synthesize ascorbic acid (AsA) from glucose, the roles of AsA in fibrotic kidney diseases are largely unknown. Unilateral ureteric obstruction (UUO) mimics the complex pathophysiology of chronic obstructive nephropathy and is an ideal model for the investigation of the roles of AsA in kidney failure. We examined the impact of a deficiency of Akr1a, a gene that encodes aldehyde reductase and is responsible for the production of AsA, on fibrotic damage caused by UUO in mice. Oxidatively modified DNA was elevated in wild-type and Akr1a-deficient kidneys as a result of UUO to a similar extent, and was only slightly suppressed by the administration of AsA. Even though Akrla-deficient mice could produce only about 10% of the AsA produced by wild-type mice, no difference was observed in collagen I synthesis under pathological conditions. The data implied either a low demand for AsA or the presence of another electron donor for collagen I production in the mouse kidney. Next, we attempted to elucidate the potential causes for oxidative damage in kidney cells during the fibrotic change. We found decreases in mitochondrial proteins, particularly in electron transport complexes, at the initial stage of the kidney fibrosis. The data imply that a dysfunction of the mitochondria leads to an elevation of ROS, which results in kidney fibrosis by stimulating cellular transformation to myofibroblasts.

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals

Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.