Slowly progressive cell death induced by GPx4-deficiency occurs via MEK1/ERK2 activation as a downstream signal after iron-independent lipid peroxidation.

Glutathione peroxidase 4 (GPx4) is an antioxidant enzyme that reduces phospholipid hydroperoxide. Studies have reported that the loss of GPx4 activity through anticancer drugs leads to ferroptosis, an iron-dependent lipid peroxidation-induced cell death. In this study, we established Tamoxifen-inducible GPx4-deficient Mouse embryonic fibroblast (MEF) cells (ETK1 cells) and found that Tamoxifen-inducible gene disruption of GPx4 induces slow cell death at ~72 h. In contrast, RSL3- or erastin-induced ferroptosis occurred quickly within 24 h. Therefore, we investigated the differences in these mechanisms between GPx4 gene disruption-induced cell death and RSL3- or erastin-induced ferroptosis. We found that GPx4-deficiency induced lipid peroxidation at 24 h in Tamoxifen-treated ETK1 cells, which was not suppressed by iron chelators, although lipid peroxidation in RSL3- or erastin-treated cells induced ferroptosis that was inhibited by iron chelators. We revealed that GPx4-deficient cell death was MEK1-dependent but RSL3- or erastin-induced ferroptosis was not, although MEK1/2 inhibitors suppressed both GPx4-deficient cell death and RSL3- or erastin-induced ferroptosis. In GPx4-deficient cell death, the phosphorylation of MEK1/2 and ERK2 was observed 39 h after lipid peroxidation, but ERK1 was not phosphorylated. Selective inhibitors of ERK2 inhibited GPx4-deficient cell death but not in RSL3- or erastin-induced cell death. These findings suggest that iron-independent lipid peroxidation due to GPx4 disruption induced cell death via the activation of MEK1/ERK2 as a downstream signal of lipid peroxidation in Tamoxifen-treated ETK1 cells. This indicates that GPx4 gene disruption induces slow cell death and involves a different pathway from RSL3- and erastin-induced ferroptosis in ETK1 cells.

Clinical Presentation of Preterm Infants with Ventricular Septal Defect.

Ventricular septal defects (VSDs) are the most common congenital heart diseases; however, case reports of preterm infants with VSD are limited. The aim of this study is to share our experience with preterm infants with VSD and to record their short-term outcomes. Between January 2000 and December 2017, 32 preterm infants with VSD were admitted to our neonatal intensive care unit at gestational age < 32 weeks. Of these, 9 were excluded by exclusion criteria. The size and location of the VSD, details of treatment, and neonatal prognosis were retrospectively reviewed from the medical records. Among the 23 preterm infants, the median gestational age was 29.4 weeks (25.0-31.3 weeks) and the median birthweight was 924 g (524-1,526 g). There were 9 infants with VSD < 2 mm and 14 infants with VSD ≥ 2 mm. For the 9 infants with VSD < 2 mm, no medical or surgical treatments for VSDs were undertaken. Of the 14 infants with VSD ≥ 2 mm, 8 (57.1%) underwent medical and surgical treatment. Surgical treatment was performed more frequently in infants with VSD ≥ 2 mm than in those with VSD < 2 mm (P = 0.007). In preterm infants, the presence of VSD ≥ 2 mm increases the risk of surgical interventions and significant patent ductus arteriosus. It is important to encourage treatment for preterm infants with VSD ≥ 2 mm, including surgical interventions, in cooperation with pediatric cardiologists.

Lipid Peroxidation-Dependent Cell Death Regulated by GPx4 and Ferroptosis.

Glutathione peroxidase 4 (Phospholipid hydroperoxide glutathione peroxidase, PHGPx) can directly reduce phospholipid hydroperoxide. Depletion of GPx4 induces lipid peroxidation-dependent cell death in embryo, testis, brain, liver, heart, and photoreceptor cells of mice. Administration of vitamin E in tissue specific GPx4 KO mice restored tissue damage in testis, liver, and heart. These results indicate that suppression of phospholipid peroxidation is essential for cell survival in normal tissues in mice. Ferroptosis is an iron-dependent non-apoptotic cell death that can elicited by pharmacological inhibiting the cystine/glutamate antiporter, system Xc- (type I) or directly binding and loss of activity of GPx4 (Type II) in cancer cells with high level RAS-RAF-MEK pathway activity or p53 expression, but not in normal cells. Ferroptosis by Erastin (Type I) and RSL3 (RAS-selective lethal 3, Type II) treatment was suppressed by an iron chelator, vitamin E and Ferrostatin-1, antioxidant compound. GPx4 can regulate ferroptosis by suppression of phospholipid peroxidation in erastin and RSL3-induced ferroptosis. Recent works have identified several regulatory factors of erastin and RSL3-induced ferroptosis. In our established GPx4-deficient MEF cells, depletion of GPx4 induce iron and 15LOX-independent lipid peroxidation at 26 h and caspase-independent cell death at 72 h, whereas erastin and RSL3 treatment resulted in iron-dependent ferroptosis by 12 h. These results indicated the possibility that the mechanism of GPx4-depleted cell death might be different from that of ferroptosis induced by erastin and RSL3.

Wakayama Medical University Hospital perinatal helicopter ambulance service: 14 year review.

BACKGROUND: In 2003, a perinatal helicopter air ambulance service was introduced for remote areas of Wakayama and Mie prefectures, Japan, but its long-term impact on perinatal medicine has not yet been analyzed. METHODS: A retrospective observational study was conducted on helicopter air ambulance cases recorded between January 2003 and December 2016 at Wakayama Medical University Hospital (WMUH). RESULTS: During that period, 61 pregnant mothers were transferred by helicopter air ambulance to WMUH. Between 2003 and 2009, the mean period from transfer to birth was 0.6 weeks, whereas between 2008 and 2016, this increased to 1.6 weeks, and the survival rate of infants born after transfer did not differ significantly (84.2%, 32/38 versus 82.1%, 23/28). Seventy-three neonates were transferred. The number transferred between 2003 and 2009 was 46, whereas this decreased to 27 between 2010 and 2016. The neonatal mortality rate in south Wakayama plus south Mie gradually decreased. The reasons for the longer period from transfer to birth, and the decrease in the rate of very low-birthweight infants after transfer may be due to development in the management of threatened premature labor, and the earlier transfer of such cases by regional obstetricians. The reasons for the decline in neonatal transfer may have included the development of fetal diagnostic techniques and improved efficiency of neonatal ground-transport in the South Wakayama region. CONCLUSION: The helicopter air ambulance is an important form of medical transportation in the south Kii peninsula.

Activation of p38 Mitogen-Activated Protein Kinase by Clotrimazole Induces Multidrug Resistance-Associated Protein 3 Activation through a Novel Transcriptional Element.

Multidrug resistance-associated protein 3 (MRP3) is a basolaterally localized transporter in the liver and contributes to the transport of various metabolites such as conjugates of endogenous compounds and drugs from hepatocytes. MRP3 expression in the human liver is low under normal physiologic conditions but is induced by drug treatment. Although several studies have identified a region necessary for the basal transcription of MRP3, no region that responds to drugs has been reported. To identify the xenobiotic-responsive elements of MRP3, we constructed a luciferase reporter plasmid containing the MRP3 5'-flanking region up to -10 kb upstream from the transcription start site. Among typical nuclear receptor ligands, clotrimazole dramatically enhanced MRP3 reporter activity in HepG2 cells, whereas rifampicin had no effect. We then conducted MRP3 reporter assays with deletion or mutation constructs to identify a clotrimazole-responsive element. The element was located approximately -6.8 kb upstream from the MRP3 transcription start site. Overexpression of the pregnane X receptor did not enhance clotrimazole-mediated transcription. We found that clotrimazole was toxic to HepG2 cells and we therefore investigated whether mitogen-activated protein kinase (MAPK) activation is involved in the transactivation of MRP3 by clotrimazole. p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] suppressed MRP3 mRNA expression induced by clotrimazole, whereas c-Jun N-terminal kinase inhibitor SP600125 (1,9-pyrazoloanthrone) and extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] did not. Phosphorylated p38 MAPK was detected in HepG2 cells treated with clotrimazole. These results suggest that activation of the p38 MAPK pathway induces the transcriptional activation of MRP3.

Development of a highly reproducible system to evaluate inhibition of cytochrome P450 3A4 activity by natural medicines.

PURPOSE: In recent years, a number of natural medicines have been reported to have inductive or inhibitive effects on the activity of drug metabolizing enzymes, upon co-administration with prescribed medicines. However, information regarding natural medicine-drug interactions that influence drug metabolism is limited owing to the lack of efficient screening method for such interactions. Therefore, to understand whether P450 activity is affected by natural medicine in small intestines, we have established frozen recombinant P450-expressing cells infected with human CYP3A4 expressing adenovirus (Ad-CYP3A4) to evaluate the effect of natural medicines on CYP3A4 activity. METHODS: Ad-CYP3A4 cells were created by infecting HepG2 cells with Ad-CYP3A4 at 10 multiplicity of infection (MOI) and these cells were stored using cryopreservation medium (fAd-CYP3A4 cells) to obtain long-term consistent data and stable supplies of cells expressing a constant level of CYP3A4 activity. RESULTS: The CYP3A4 activity in fAd-CYP3A4 cells remained unaffected at the end of each frozen period (0, 1, 2, and 6 months). Inhibitory effect on CYP3A4 activity by typical inhibitors (ketoconazole, hyperforin) and natural medicines (Cat's Claw, Devil's Claw, Feverfew, Peppermint Oil, Red Clover, and Siberian Eleuthero) were evaluated. The inhibitors had nearly equal IC50 values in fAd-CYP3A4 cells, Ad-CYP3A4 cells and recombinant CYP3A4 microsomes. Cat's Claw, Peppermint Oil and Siberian Eleuthero inhibited CYP3A4 activity more potently than 0.1 µM ketoconazole in fAd-CYP3A4 cells. CONCLUSIONS: In the present study, we have successfully developed a highly reproducible system to evaluate CYP3A4 inhibition in small intestines by natural medicines.

Simultaneous evaluation of human CYP3A4 and ABCB1 induction by reporter assay in LS174T cells, stably expressing their reporter genes.

The bioavailability of orally administered therapies are often significantly limited in the human intestine by the metabolic activities of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). Predicting whether candidate compounds induce CYP3A4 and P-gp is a crucial stage in the drug development process, as drug-drug interactions may result in the induction of intestinal CYP3A4 and P-gp. However, the assay systems needed to evaluate both CYP3A4 and P-gp induction in the intestine are yet to be established. To address this urgent requirement, LS174T cells were used to create two stable cell lines expressing the CYP3A4 or ATP-binding cassette subfamily B member 1 (ABCB1, encoding P-gp) reporter genes. First, these stable cells were tested by treatment with 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis RA) that induce CYP3A4 and P-gp in the intestines. All these compounds significantly increased both CYP3A4 and ABCB1 reporter activities in the stable cell lines. To simultaneously assess the induction of CYP3A4 and ABCB1, both stable cells were co-cultivated to measure their reporter activities. The mixed cells showed a significant increase in the CYP3A4 and ABCB1 reporter activities following treatment with 1,25(OH)2 D3, ATRA, and 9-cis RA. These activity levels were maintained after passaging more than 20 times and following multiple freeze-thaw cycles. These results demonstrate that our established cell lines can be used to evaluate simultaneously CYP3A4 and ABCB1 induction in the intestines, providing a valuable in vitro model for the evaluation of future drug candidates.

Adverse perinatal and neonatal outcomes in patients with chronic abruption-oligohydramnios sequence.

AIM: Chronic abruption-oligohydramnios sequence (CAOS) is a clinical condition with lasting vaginal bleeding and oligohydramnios because of chronic placental abruption, which seems to cause preterm labor and neonatal chronic lung disease (CLD). This prompted us to explore the correlation between perinatal/neonatal outcomes and CAOS. METHODS: Patients with suspected risk of abortion with recurrent vaginal bleeding were divided into CAOS and non-CAOS groups, and we compared the perinatal/neonatal outcomes between these two groups. To examine the impact of chorioamnionitis (CAM) on the prognosis of CAOS, we also compared outcomes between the CAOS group and gestational-age-matched preterm labor due to CAM (CAM group). RESULTS: In the CAOS and non-CAOS groups, initial vaginal bleeding was seen at the first trimester. However, its duration was significantly longer in the CAOS group. Additionally, neonatal birthweight was lower, and small-for-gestational-age (SGA) incidence was higher in the CAOS group. CLD was observed in most infants from CAOS patients. In the comparison between CAOS and CAM groups, birthweight was lower and SGA incidence was higher in the CAOS group. Moreover, CLD incidence and neonatal mortality were significantly higher, despite the lower incidence of severe CAM in the CAOS group. Finally, multivariate analysis demonstrated that duration of bleeding was a significant predictive factor for CAOS. CONCLUSIONS: Our observations demonstrated that patients with CAOS were a high-risk group for poor perinatal/neonatal outcomes. Moreover, episodes of recurrent and prolonged uterine bleeding were predictive factors for CAOS. During the first trimester, prolonged bleeding is an important sign as one symptom of CAOS.

A synthetic biotinylated peptide, BP21, inhibits the induction of mRNA expression of inflammatory substances by oxidized- and lyso-phosphatidylcholine.

Preclinical Research Oxidized low-density lipoprotein (ox-LDL) is implicated in many inflammatory diseases, e.g., type 2 diabetes, obesity, atherosclerosis, and metabolic syndrome. We previously reported that a synthetic biotinylated peptide, BP21, inhibits the bioactivity of ox-LDL via direct binding to ox-LDL. Here, we investigated the effect of BP21 on the mRNA expression of proinflammatory mediators induced by two major components of ox-LDL, oxidized- and lyso-phosphatidylcholine (ox-PC and LPC), in monocytes/macrophages (THP-1 cells) and adipocytes (3T3-L1 cells). In THP-1 cells, BP21 markedly reduced the mRNA expression of interleukin (IL)-6, adipocyte fatty acid-binding protein (aP2), tumor necrosis factor-α, and mitogen-activated protein kinase phosphatase-1, which are induced by one of the major bioactive components of ox-PC, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), and reduced the mRNA expression of IL-6, the ox-LDL-specific scavenger receptor CD36, and aP2 induced by LPC. In adipocytes, the mRNA expression of IL-1ß as an adipokine and aP2 is highly induced by ox-PC and LPC, and BP21 markedly reduced the mRNA expression of IL-1ß and aP2 induced by POVPC and LPC. Furthermore, BP21 specifically bound to LPC and POVPC in a dose-dependent manner. These results suggest that BP21 may be useful lead for the potential treatment and prevention of inflammatory diseases caused by ox-PC and LPC.

Exploring Acute Liver Damage: Slimming Health Foods and CYP3A4 Induction.

Background: Patients taking multiple drugs and various health foods often develop acute hepatitis. We hypothesized that the interaction between health foods and drug metabolism was the cause of severe liver injury in these patients. Therefore, we studied changes in the activity of the drug-metabolizing enzyme, cytochrome P450 (CYP), using slimming health food extracts and elucidated the molecular mechanism of liver injury onset through hepatotoxicity evaluation. Methods: For cytotoxicity testing, health food extract samples were added to HepG2 cells derived from hepatic parenchymal cells and culture medium, and cell viability was calculated 48 h after culture. To evaluate CYP3A4 induction, 3-1-10 cells constructed with a reporter linked to CYP3A4 gene were used, and reporter activity was measured 48 h after culture. Results: In the chronological order of the slimming health food intake history of the patient, niacinamide and Gymnema sylvestre extracts strongly inhibited HepG2 cell viability. In contrast, dietary supplements A and Coleus forskohlii extract strongly induced CYP3A4 reporter activity.To confirm CYP3A4 induction in humans, humanized CYP3A/pregnane X receptor (PXR) mice were treated with forskolin. CYP3A4 mRNA expression levels were elevated 3.9 times compared to that of the control group (P < 0.05). Conclusion: Coleus forskohlii extract showed the strongest transcriptional activation of CYP3A4 gene. In a mouse model of human-type drug metabolism, forskolin induced CYP3A4 transcription. Thus, we concluded that CYP3A4 induction by Coleus forskohlii is one of the causes of crucial hepatocellular injury, which is a type of liver injury caused by the active metabolite of acetaminophen produced by CYP3A4.

Mechanism Underlying Conflicting Drug-Drug Interaction Between Aprepitant and Voriconazole via Cytochrome P450 3A4-Mediated Metabolism.

Background: Voriconazole is an antifungal drug for which therapeutic monitoring is recommended to prevent side effects. Temporary administration of the antiemetic drug fosaprepitant remarkably decreases the plasma concentration of voriconazole from the therapeutic range. The ratio of the major metabolite voriconazole N-oxide to voriconazole exceeded that at any other time for a patient who started chemotherapy during voriconazole therapy. We attributed this unpredictable result to cytochrome P450 3A4 induced by aprepitant that was converted from fosaprepitant in vivo. Methods: Concentrations of voriconazole and voriconazole N-oxide were measured using liquid chromatography-mass spectrometry/mass spectrometry in primary human hepatocytes after incubation with aprepitant. Aprepitant suppressed voriconazole N-oxide formation within 24 h, followed by a continuous increase. Levels of drug-metabolizing cytochrome P450 mRNA were measured using real-time PCR in primary human hepatocytes incubated with aprepitant. Results: Cytochrome P450 3A4 and 2C9 mRNA levels increased ~4- and 2-fold, respectively, over time. Cytochrome P450 3A4 induction was confirmed using reporter assays. We also assessed L-755446, a major metabolite of aprepitant that lacks a triazole ring. Both compounds dose-dependently increased reporter activity; however, induction by L-755446 was stronger than that by aprepitant. Conclusion: These results indicate that aprepitant initially inhibited voriconazole metabolism via its triazole ring and increased cytochrome P450 3A4 induction following L-755446 formation. The decrease in plasma voriconazole concentration 7 days after fosaprepitant administration was mainly attributed to cytochrome P450 3A4 induction by L-755446.

Acidic sphingomyelinase induced by electrophiles promotes proinflammatory cytokine production in human bladder carcinoma ECV-304 cells.

Electrophiles in environmental pollutants or cigarette smoke are high risk factors for various diseases caused by cell injuries such as apoptosis and inflammation. Here we show that electrophilic compounds such as diethyl malate (DEM), methyl mercury and cigarette smoke extracts significantly enhanced the expression of acidic sphingomyelinase (ASMase). ASMase activity and the amount of ceramide of DEM-treated cells were approximately 6 times and 4 times higher than these of non-treated cells, respectively. Moreover, we found that DEM pretreatment enhanced the production of IL-6 induced by TNF-α. Knockdown of ASMase attenuated the enhancement of TNF-α-dependent IL-6 production. On the other hand, enhancement of TNF-α-induced IL-6 production was observed in ASMase-overexpressing cells without DEM. Fractionation of the lipid raft revealed that the TNF receptor 1 (TNFR1) was migrated into the lipid raft in DEM-treated cells or ASMase-overexpressing cells. The TNF-α-induced IL-6 expression required the clustering of TNFR1 since IL-6 expression were decreased by the destruction of the lipid raft with filipin. These results demonstrated a new role for ASMase in the acceleration of the production of TNF-induced IL-6 as a pro-inflammatory cytokine and indicated that electrophiles could potentiate inflammation response by up-regulating of ASMase expression following formation of lipid rafts.

Development of an adenovirus-mediated reporter assay system to detect a low concentration of retinoic acid in MCF-7 cells.

Retinoic acid, an active form of vitamin A, plays very important roles in mammalian embryogenesis. The concentration of retinoic acid is extremely low and strictly regulated by enzymes of cytochrome P450 (CYP) family, CYP26s (CYP26A1, CYP26B1 and CYP26C1) in the cells. Therefore, it is thought that changes in CYP26s activities due to exposure to a wide variety of drugs and chemicals exhibit teratogenicity. In this study, to easily detect the changes in retinoic acid level, we constructed an adenovirus-mediated reporter assay system using the promoter region of the CYP26A1 gene and inserting retinoic acid response element (RARE) and retinoid X response element (RXRE) into the downstream of the luciferase gene of reporter plasmid, which highly increased the response to retinoic acid. Reporter activity significantly increased in a concentration-dependent manner with retinoic acid; this increase was also observed at least after treatment with a very low concentration of 1 nM retinoic acid. This increase was suppressed by the accelerated metabolism of retinoic acid due to the overexpression of CYP26A1; however, this suppression was almost completely suspended by treatment with talarozole, a CYP26 inhibitor. In conclusion, the reporter assay system constructed using the induction of CYP26A1 expression is a risk assessment system that responds to extremely low concentrations of retinoic acid and is useful for assessing the excess vitamin A mediated teratogenicity caused by various chemicals at the cellular level.

Changes in maternal and child health outcomes after introduction of a helicopter into perinatal transportation in Japan.

To examine the effect of perinatal helicopter transportation on maternal and child health. Helicopter transportation for the sparsely populated southern areas of Wakayama and Mie prefectures was introduced in June 2003. Maternal and child health statistics for 2000-2002 and 2004-2006 were compared between the south and north regions of the prefectures. There were 9 maternal transports from south Wakayama, 2 from north Wakayama, and 5 from south Mie in 2004-2006; and 13 neonatal transports from south Wakayama and 7 from north Wakayama during the same period. Decreases in neonatal and perinatal mortalities in 2004-2006 compared to 2000-2002 were greater in south Wakayama than in north Wakayama (-0.31 vs. -0.28, and -0.57 vs. -0.18, respectively); and greater in south Mie than in north Mie (-0.90 vs. -0.49, and -2.49 vs. -1.48, respectively). The changes in the number of maternal deaths between 2000-2002 and 2004-2006 were 0 in south Wakayama, 1 in north Wakayama, -2 in south Mie, and -1 in north Mie, with the greatest change occurring in south Mie. Use of a helicopter for perinatal transportation can possibly improve maternal and child health in sparsely populated areas far away from urban areas.

Optimum therapeutic strategy for meconium-related ileus in very-low-birth-weight infants.

BACKGROUND/PURPOSE: therapeutic strategy for meconium-related ileus (MRI) in very-low-birth-weight infants (VLBWs) has not been established. This study aims to clarify the optimum therapeutic strategy for MRI in VLBWs. METHODS: MRI was defined as delayed meconium excretion and microcolon on contrast enema with Gastrografin (diatrizoate acid). Forty-two infants with MRI were treated at our institution between 2009 and 2019, and are reviewed here. They were classified into two groups: in group A (n=21), Gastrografin regurgitated into the dilated intestine during the first or second round of Gastrografin enema (GaE), while in group B (N = 21), Gastrografin did not regurgitate. Laparotomy was indicated if the intestine was perforated, or if abdominal distention was not relieved by two rounds of GaE. RESULTS: in group A, meconium was excreted in all cases within 24 h after GaE, and no cases required laparotomy. In group B, twelve cases (57%) underwent laparotomy (P < 0.01), six cases in this group (29%), showed free air on X-ray images (P < 0.01). The median hospital stay in groups A and B were 89.0 and 136.5 days, respectively (P < 0.05). Overall mortality was 2.4%. CONCLUSIONS: early therapeutic diagnosis by GaE followed by early surgery is suggested as the optimum strategy for MRI in VLBWs.

Involvement of independent mechanism upon poly(ADP-ribose) polymerase (PARP) activation in methylmercury cytotoxicity in rat cerebellar granule cell culture.

Poly(ADP-ribose) polymerase (PARP) activation plays a role in repairing injured DNA, while its overactivation is involved in various diseases, including neuronal degradation. In the present study, we investigated the use of a PARP inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), whether methylmercury-induced cell death in the primary culture of cerebellar granule cells involved PARP activation. DPQ decreased the methylmercury-induced cell death in a dose-dependent manner. Unexpectedly, this protective effect was DPQ specific; none of the other PARP inhibitors--1,5-dihydroxyisoquinoline, 3-aminobenzamide, or PJ34--affected neuronal cell death. Methylmercury-induced cell death involves the decrease of glutathione (GSH) and production of reactive oxygen species. Therefore, to understand the mechanism by which DPQ inhibits cytotoxicity, we first studied the effect of DPQ on buthionine sulfoximine- or diethyl maleate-induced death of primary cultured cells and human neuroblastoma IMR-32 cells, both of which are mediated by GSH depletion. DPQ inhibited the cell death of both cultured cells, but it did not restore the decrease of cellular GSH by buthionine sulfoximine to the control level. Second, we evaluated the antioxidant activity of PARP inhibitors by methods with ABTS (2-2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) or DPPH (1,1-diphenyl-2-picrylhydrazyl) used as a radical because antioxidants also efficiently suppress methylmercury-induced cell death. The antioxidant activity of DPQ was the lowest among the tested PARP inhibitors. Taken together, our results indicate that DPQ effectively protects cells against methylmercury- and GSH depletion-induced death. Furthermore, they suggest that DPQ exerts its protective effect through a mechanism other than PARP inhibition and direct antioxidation, and that PARP activation is not involved in methylmercury-induced neuronal cell death.

The common function of a novel subfamily of B-Box zinc finger proteins with reference to circadian-associated events in Arabidopsis thaliana.

Over 1,600 genes encoding putative transcription factors have been identified in the Arabidopsis genome sequence, however, their physiological functions are not yet fully understood. In this study, a small subfamily of double B-box zinc finger (DBB, DOUBLE B-BOX) genes, encoding eight putative transcription factors, were characterized with reference to the circadian rhythm and the early photomorphogenic regulation of hypocotyl elongation in response to light signals. Among these, it was found that the transcriptions of five DBB genes were under the control of circadian rhythm. To gain insight into the physiological roles of these putative transcription factors, forward and reverse genetic studies were carried out. The results suggested that they are commonly implicated in light signal transduction during early photomorphogenesis, however, their functions are not totally redundant, as judged by the fact that their circadian-expression profiles (or phases) were distinctive from each other, and by the fact that some DBBs (named DBB1a, DBB1b, STO, and STH) were apparently implicated in light signal transduction in a negative manner, whereas another (named DBB3) was implicated in a positive manner with regard to light-induced inhibition of elongation of hypocotyls. We also found that homologous B-box zinc finger genes are widely conserved in higher plants (e.g., Oryza sativa). Taking this altogether, it is probable that in addition to previously characterized bZIP-type transcription factors (e.g., HY5 and HYH) and bHLH-type transcription factors (e.g., PIF4 and PIF5/PIL6), a set of B-box zinc finger transcription factors should also be taken into consideration for a better understanding of the complex molecular mechanisms underlying the early photomorphogenic development of Arabidopsis thaliana.

Effect of health foods on cytochrome P450-mediated drug metabolism.

BACKGROUND: Health foods have been widely sold and consumed in Japan. There has been an increase in reports of adverse effects in association with the expanding health food market. While health food-drug interactions are a particular concern from the viewpoint of safe and effective use of health foods, information regarding such interactions is limited owing to the lack of established methods to assess the effects of health food products on drug metabolism. We therefore developed cells that mimicked the activities of cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4, which strongly contribute to drug metabolism in human hepatocytes, and established a system to assess the inhibitory activity of health foods toward P450-mediated metabolism. METHODS: We simultaneously infected HepG2 cells with five P450-expressing adenoviruses (Ad-CYP1A2, Ad-CYP2C9, Ad-CYP2C19, Ad-CYP2D6, and Ad-CYP3A4) to mimic the activity levels of these P450s in human hepatocytes, and named them Ad-P450 cells. The activity levels of P450s in Ad-P450 cells and human hepatocytes were calculated via simultaneous liquid chromatography/tandem mass spectrometry analysis utilizing a P450 substrate cocktail. RESULTS: We established Ad-P450 cells mimicking the activity levels of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human hepatocytes. We determined the Km values of P450 substrates and IC50 values of P450 inhibitors in Ad-P450 cells. These values were approximately equivalent to those obtained in previous studies. We investigated the inhibitory effects of 172 health foods that were recently in circulation in Japan on P450-mediated metabolism using Ad-P450 cells. Of the 172 health foods, five products (two products having dietary effects, one turmeric-based product, one collagen-based product, and one propolis-containing product) simultaneously inhibited the five P450s by more than 50%. Another 29 products were also confirmed to inhibit one or more P450s. CONCLUSIONS: We established a comprehensive assessment system to elucidate the effects of health foods on P450-mediated metabolism and identified the inhibitory activity of 34 of 172 health foods toward the drug-metabolizing P450s. Our results may provide useful information to predict health food-drug interactions.

Defects in autophagosome-lysosome fusion underlie Vici syndrome, a neurodevelopmental disorder with multisystem involvement.

Vici syndrome (VICIS) is a rare, autosomal recessive neurodevelopmental disorder with multisystem involvement characterized by agenesis of the corpus callosum, cataracts, cardiomyopathy, combined immunodeficiency, developmental delay, and hypopigmentation. Mutations in EPG5, a gene that encodes a key autophagy regulator, have been shown to cause VICIS, however, the precise pathomechanism underlying VICIS is yet to be clarified. Here, we describe detailed clinical (including brain MRI and muscle biopsy) and genetic features of nine Japanese patients with VICIS. Genetic dissection of these nine patients from seven families identified 14 causative mutations in EPG5. These included five nonsense, two frameshift, three splicing, one missense, and one multi-exon deletion mutations, and two initiation codon variants. Furthermore, cultured skin fibroblasts (SFs) from two affected patients demonstrated partial autophagic dysfunction. To investigate the function of EPG5, siRNA based EPG5 knock-down, and CRISPR/Cas9 mediated EPG5 knock-out HeLa cells were generated. EPG5-depleted cells exhibited a complete block of autophagic flux caused by defective autophagosome-lysosome fusion. Unexpectedly, endocytic degradation was normal in both VICIS SFs and EPG5 depleted cells, suggesting that EPG5 function is limited to the regulation of autophagosome-lysosome fusion.