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Some mutations of isocitrate dehydrogenase 1 and 2 observed in multiple kinds of malignant tumors can lead to a neomorphic enzyme activity that converts alpha-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). As an oncometabolite, 2-HG can cause epigenetic changes and impair cell differentiation. Inhibiting the activity of isocitrate dehydrogenase mutants (mIDH) is considered to be an effective therapy for the treatment of mIDH positive cancers, including glioma and acute myeloid leukemia (AML). The presently disclosed allosteric inhibitors work only on one of the mIDH1 and mIDH2, and it is shown that mIDH1 and mIDH2 have different allosteric inhibition pockets. However, AG-881 from Agios Pharmaceuticals was found to be a pan-IDH inhibitor against both mIDH1 and mIDH2, and is undergoing Phase I clinical trials for tumors with an IDH1 and/or IDH2 mutation. To understand the binding mode of AG-881 to mIDHs, we solved the crystal structures of IDH1-R132H/NADPH/AG-881 and IDH2-R140Q/NADPH/AG-881 complexes, and acquired the IC 50 values of AG-881 for IDH1-R132H and IDH2-R140Q homodimers after different pre-incubation times. Our data show that AG-881 binds IDH1-R132H and IDH2-R140Q in the same allosteric pockets and that the subtle difference in the pockets of these two proteins may contribute to their remarkably different inhibitory kinetics by AG-881. The structural pharmacological data provided in this report may benefit the future development of pan-IDH inhibitors targeting mIDH1 and mIDH2., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lung cancer is the leading cause of cancer deaths. Epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small cell lung cancers (NSCLCs), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the anti-EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib initially, but soon develop resistance to them in about half of the cases due to the emergence of the gatekeeper mutation T790M. The third-generation TKIs such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to the EGFR kinase through Cys 797, but ultimately lose their efficacy upon emergence of the C797S mutation that abolishes the covalent bonding. Therefore to develop new TKIs to overcome EGFR drug-resistant mutants harboring T790M/C797S is urgently demanded. EAI001 and EAI045 are a new type of EGFR TKIs that bind to EGFR reversibly and not relying on Cys 797. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR L858R/T790M and L858R/T790M/C797S. Here we report the crystal structure of EGFR T790M/C797S/V948R in complex with EAI045, and compare it to EGFR T790M/V948R in complex with EAI001. The complex structure reveals why EAI045 binds tighter to EGFR than does EAI001, and why EAI001 and EAI045 prefer binding to EGFR T790M. The knowledge may facilitate future drug development studies targeting this very important cancer target., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Drug-resistance is a major challenge in targeted therapy of EGFR mutated non-small cell lung cancers (NSCLCs). The third-generation irreversible inhibitors such as AZD9291, CO-1686 and WZ4002 can overcome EGFR T790M drug-resistance mutant through covalent binding through Cys 797, but ultimately lose their efficacy upon emergence of the new mutation C797S. To develop new reversible inhibitors not relying on covalent binding through Cys 797 is therefore urgently demanded. Gö6976 is a staurosporine-like reversible inhibitor targeting T790M while sparing the wild-type EGFR. In the present work, we reported the complex crystal structures of EGFR T790M/C797S + Gö6976 and T790M + Gö6976, along with enzyme kinetic data of EGFR wild-type, T790M and T790M/C797S. These data showed that the C797S mutation does not significantly alter the structure and function of the EGFR kinase, but increases the local hydrophilicity around residue 797. The complex crystal structures also elucidated the detailed binding mode of Gö6976 to EGFR and explained why this compound prefers binding to T790M mutant. These structural pharmacological data would facilitate future drug development studies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Streptomyces avermitilis is an actinobacterium known to produce clinically useful macrolides including avermectins. CYP107L2 from S. avermitilis shares a high sequence similarity with the PikC (CYP107L1) from S. venezuelae. To elucidate the structural features of CYP107L2, we conducted biochemical and structural characterization of CYP107L2 from S. avermitilis. The CYP107L2 gene was cloned, and its recombinant protein was expressed and purified. The CYP107L2 showed a low-spin state of heme, and the reduced form yielded the CO difference spectra with a maximal absorption at 449 nm. Binding of pikromycin and lauric acid yielded the typical type I spectra with K d values of 4.8 ± 0.3 and 111 ± 9 μM, respectively. However, no metabolic product was observed in the enzyme reaction. X-ray crystal structures of the ligand-free CYP107L2 and its complex with lauric acid were determined at the resolution of 2.6 and 2.5 Å, respectively. CYP107L2 showed a well-conserved CYP structure with a wide-open substrate-binding cavity. The lauric acid is bound mainly via hydrophobic interactions with the carboxylate group of lauric acid coordinated to the heme of P450. Glu-40 and Leu-382 residues in the CYP107L2 complex with lauric acid showed significant conformational changes to provide plentiful room for the lauric acid in the substrate-binding site., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Platelet-derived growth factor receptor α (PDGFRA) is a Type III receptor tyrosine kinase, and this kinase is a target for treatment of gastrointestinal stromal tumors (GIST) as it is frequently mutated in these cancers. Most of the mutations that cause constitutive activation of PDGFRA occur in either the activation loop (A-loop) or in the juxtamembrane (JM) domain, such as the mutations D842V or V561D respectively. Treatment of PDGFRA-mutated GIST with imatinib is successful in some cases, but the D842V mutation is imatinib-resistant. To better understand the mechanism of PDGFRA drug-resistance, we have determined the crystal structure of the PDGFRA kinase domain in the auto-inhibited form, and studied the kinetics of the D842V mutation. Auto-inhibited PDGFRA is stabilized by the JM domain, which inserts into the active site of the kinase. The conserved residue Asp842 makes extensive contacts with several A-loop residues to maintain PDGFRA in the "DFG out" conformation, which stabilizes the kinase in the inactive state and facilitates the binding of imatinib. The D842V mutation would therefore be expected to activate the kinase and hinder the binding of drug through destabilizing the "DFG out" conformation. Furthermore, our kinetic data show that drug resistance in the D842V mutation may also in part result from its increased affinity for ATP. The PDGFRA kinase domain structure we report in this study has potential to facilitate development of new agents which can inhibit this kinase, including both its activating and drug-resistant mutations., (Copyright © 2016. Published by Elsevier Inc.)

Vascular calcification is a hallmark of cardiovascular disease. Interleukin-24 (IL-24) has been known to suppress tumor progression in a variety of human cancers. However, the role of IL-24 in the pathophysiology of diseases other than cancer is unclear. We investigated the role of IL-24 in vascular calcification. IL-24 was applied to a β-glycerophosphate (β-GP)-induced rat vascular smooth muscle cell (VSMC) calcification model. In this study, IL-24 significantly inhibited β-GP-induced VSMC calcification, as determined by von Kossa staining and calcium content. The inhibitory effect of IL-24 on VSMC calcification was due to the suppression of β-GP-induced apoptosis and expression of calcification and osteoblastic markers. In addition, IL-24 abrogated β-GP-induced activation of the Wnt/β-catenin pathway, which plays a key role in the pathogenesis of vascular calcification. The specificity of IL-24 for the inhibition of VSMC calcification was confirmed by using a neutralizing antibody to IL-24. Our results suggest that IL-24 inhibits β-GP-induced VSMC calcification by inhibiting apoptosis, the expression of calcification and osteoblastic markers, and the Wnt/ β-catenin pathway. Our study may provide a novel mechanism of action of IL-24 in cardiovascular disease and indicates that IL-24 is a potential therapeutic agent in VSMC calcification., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Krüppel-like factor 10 (KLF10) has been suggested to be a putative tumor suppressor. In the present study, we generated KLF10 deficient mice to explore this hypothesis in vivo. KLF10 deficient mice exhibited increased predisposition to skin tumorigenesis and markedly accelerated papilloma development after DMBA/TPA treatment. On the other hand, KLF10 deficient keratinocytes showed increased proliferation and apoptosis. In colony formation assays after oncogenic H-Ras transfection, KLF10 deficient mouse embryonic fibroblasts (MEFs) yielded more colonies than wild-type MEFs. Furthermore, KLF10 dose-dependently activated p21(WAF1/CIP1) transcription, which was independent of p53 and Sp1 binding sites in p21(WAF1/CIP1) promoter. This study demonstrates that KLF10 is a tumor suppressor and that it targets p21(WAF1/CIP1) transcription., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Oncostatin M (OSM) is a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family that has been found to be involved in both pro- and anti-inflammatory responses in cell-mediated immunity. Maturation of dendritic cells (DCs) is crucial for initiation of primary immune responses and is regulated by several stimuli. In this study, the role of OSM in the phenotypic and functional maturation of DCs was evaluated in vitro. Stimulation with OSM upregulated the expression of CD80, CD86, MHC class I and MHC class II and reduced the endocytic capacity of immature DCs. Moreover, OSM induced the allogeneic immunostimulatory capacity of DCs by stimulating the production of the Th1-promoting cytokine IL-12. OSM also increased the production of IFN-gamma by T cells in mixed-lymphocyte reactions, which would be expected to contribute to the Th1 polarization of the immune response. The expression of surface markers and cytokine production in DCs was mediated by both the MAPK and NF-kappaB pathways. Taken together, these results indicate that OSM may play a role in innate immunity and in acquired immunity by enhancing DCs maturation and promoting Th1 immune responses., (2010 Elsevier Inc. All rights reserved.)

Alpha-eleostearic acid (alpha-ESA, 9Z11E13E-18:3), a linolenic acid isomer with a conjugated triene system, is a natural and biologically-active compound that has been shown to possess potent anti-tumor properties. Herein, we demonstrate alpha-ESA induced apoptosis and autophagy with reactive oxygen species (ROS) generation in HeLa cells. Treatment with alpha-ESA caused inhibition of phosphorylated (p)AKT and elongated the sub G1 phase in the cell cycle, indicating induction of apoptosis. Autophagy was also induced by alpha-ESA treatment, causing low pAKT and pP70S6K activities, increasing pERK1/2 and leading to a higher conversion rate of LC3 I to LC3 II compared to that of the control. The autophagy was further confirmed by fluorescence microscopy and flow cytometry through monodansylcadavarine (MDC) staining. It appears that the role of autophagy is a protective mechanism against cell death in alpha-ESA-treated HeLa cells. Subsequently, we found that treating HeLa cells with alpha-ESA induced the generation of reactive oxygen species (ROS). The phosphorylation of P70S6K, downstream of mTOR signaling, and AKT were further reduced by pretreatment with N-acetyl-l-cysteine (NAC), an ROS scavenger, whereas the phosphorylation of ERK1/2 and the conversion of LC3 I to LC3 II were further enhanced. As a result, the blocking of the action of ROS promoted alpha-ESA-induced apoptosis and autophagy. Taken together, our results indicate that the generation of ROS by alpha-ESA treatment impedes the progress of apoptosis and excessive autophagy formation which takes part in cell death, thus impeding death promotion., (Copyright 2009 Elsevier Inc. All rights reserved.)

We have previously reported that TTF-1, a homeodomain-containing transcription factor, regulates circadian rhythm of pituitary adenylate cyclase-activating polypeptide gene expression in the rat hypothalamus. In this study we found that TTF-1 mRNA was specifically expressed in the rat suprachiasmatic nucleus (SCN) and colocalized with Period 2 (Per2), a circadian feedback loop controller. Interaction between TTF-1 and Per1 and Per2 was demonstrated by immunoprecipitation and immunoblot assays. Moreover, TTF-1 and Per proteins additively stimulated a transcriptional activity of angiotensinogen (AoGen) gene. TTF-1 also activated in vitro rhythm of AoGen transcription determined by secretary alkaline phosphatase (SEAP) reporter system in the NIH3T3 cells. These results suggest that TTF-1 plays a role in the circadian rhythm regulation of the AoGen gene expression via interacting with Per proteins in the rat SCN.

Cidec is a lipid droplet-associated protein, which inhibits lipolysis, leading to the accumulation of triglycerides in adipocytes. However, the transcriptional regulation of Cidec in adipocyte remains unknown. In the present study we investigated that the mouse Cidec transcript is regulated by PPARgamma2. After the differentiation of adipocyte, the expression pattern of Cidec was similar to that of PPARgamma2. In the presence of a PPARgamma agonist, the level of Cidec mRNA was highly increased. In addition, putative PPRE sites were identified in the Cidec promoter. By chromatin immunoprecipitation assay and reporter assay, we observed the binding of PPARgamma2 to the promoter of Cidec. Gel shift assay and the mutagenesis study were showed that the -219/-207 region of the Cidec promoter could function as a PPRE of the Cidec promoter. These results suggest that PPARgamma2 is required for the transcriptional activity of Cidec during adipogenesis, which could be contributed to understand the molecular mechanism of lipid droplet formation in adipocytes.

Interaction of human cytochrome P450 1A2 (CYP1A2) and 2E1 (CYP2E1) with phospholipid, lysophosphatidylserine (LysoPS) in the context of a PC matrix specifically stimulated the PLD activity of both enzymes in a LysoPS concentration-dependent manner. However, other anionic lysophospholipids as well as the neutral lysophospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine, had no effect. LysoPS also accompanied conformational changes in both CYPs when assayed by circular dichroism. Although the PLD activity was decreased in the presence of components required for the monooxygenase (MMO) activity, including 100% PC membranes, NADPH-cytochrome P450 reductase and NADPH, as compared to the activity in the absence of the reducing system, LysoPS recovered the PLD activity in a concentration-dependent manner. Considering that LysoPS induced a decrease in the MMO activities of both CYPs, the results suggest that the functional roles of CYP1A2 and 2E1 can be switched by interaction with a specific anionic lysophospholipid in vivo.

Resistin is an adipokine whose physiologic role in obesity, type II diabetes mellitus, and inflammatory diseases has been a subject of debate because while it is expressed in adipocytes and adipose tissue in mouse, it is expressed in leukocytes, such as macrophages, in human. In the present study, we attempt to define the effect of resistin on human dendritic cells (DCs) derived from CD14(+) monocytes. When DCs were stimulated with lipoteichoic acid (LTA) and treated with various concentrations of resistin, antigen-uptake process and the endocytic capacity of DCs were decreased. It is intriguing that resistin attenuated cytokine production in LTA-primed DCs. Consequently, T cell activity was reduced when lymphocytes were mixed with Staphylococcus aureus-primed autologous DCs treated with resistin compared to S. aureus-primed DCs without resistin. Our results suggest that resistin interferes with the efficacy of immune responses activated by Gram-positive bacterial infection in human DCs.

Thyroid transcription factor-1 (TTF-1) belongs to the Nkx family of homeodomain-containing proteins and regulates expression of several important genes in the brain. Our previous studies showed that TTF-1 plays an important role in water homeostasis in the subfornical organ of rats and is involved in cerebrospinal fluid formation by regulation of aquaporin-1 transcription in the choroid plexus. In this study, we examined changes in TTF-1 transcription in response to hypertonicity using promoter assays. TTF-1 was synthesized in several osmosensitive regions of the rat brain. TTF-1 promoter activity was diminished by treatment with hypertonic solutions in a time- and dose-dependent manner in brain-derived cell lines. Additionally, TTF-1 was involved in the regulation of angiotensinogen (Aogen) transcription under a hyperosmotic condition through specific binding domains in the Aogen promoter. These results suggest a possible role of TTF-1 in brain fluid homeostasis in response to changes in the osmotic environment.

Thyroid transcription factor 1 (TTF-1) is required for morphogenesis of the fetal diencephalon. Previous reports showed that mice carrying a TTF-1 null mutation lacked normal development of the pituitary gland. In this study, a role for TTF-1 in the regulation of growth hormone and prolactin transcription was identified. In-situ hybridization analysis demonstrated TTF-1 mRNA in the growth hormone-producing cells and prolactin-producing cells of the rat anterior pituitary gland. In the GH3 pituitary cell line, we identified TTF-1 as a factor functionally regulating growth hormone and prolactin transcription. TTF-1 activated prolactin transcription, but inhibited growth hormone transcription. Inhibition and activation of growth hormone and prolactin transcription, respectively, by TTF-1 disappeared upon deletion of the TTF-1 binding motifs within the promoters of these genes. These data suggest that TTF-1 plays a regulatory role in the transcription of growth hormone and prolactin genes and may regulate transdifferentiation of cells expressing these two hormones.

TTF-1 is a member of the NKx family of homeodomain genes, and is required for morphogenesis and fetal diencephalon development. Our previous studies have shown that TTF-1 expression is maintained in some regions of the postnatal rat brain and transactivates the gene expression of several neuropeptides. In this study, a potential role for TTF-1 in the regulation of feeding behavior was identified. Immunohistochemical analysis showed that TTF-1 is present in several hypothalamic nuclei of the adult rat brain involved in the control of feeding behavior. Food deprivation for two days markedly increased the hypothalamic levels of TTF-1 mRNA and protein. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased TTF-1 protein abundance in the hypothalamus. This TTF-1 decrease was followed by a significant decrease in neuropeptide Y mRNA content and an increase in proopiomelanocortin mRNA content, and in turn resulted in a decrease of the animal's food intake and body weight. These results suggest a novel role for TTF-1 in the regulation of feeding behavior in the rat hypothalamus.

Cyclophilins (CyPs) are a widespreading protein family in living organisms and possess the activity of peptidyl-prolyl cis-trans isomerase (PPIase), which is inhibited by cyclosporin A (CsA). The human nuclear cyclophilin (hCyP33) is the first protein which was found to contain two RNA binding domains at the amino-terminus and a PPIase domain at the carboxyl-terminus. We isolated the hCyP33 gene from the human hematopoietic stem/progenitor cells and expressed it in Escherichia coli, and determined the crystal structure of the C domain of hCyP33 at 1.88 A resolution. The core structure is a beta-barrel covered by two alpha-helices. Superposition of the structure of the C domain of hCyP33 with the structure of CypA suggests that the C domain contains PPIase active site which binds to CsA. Furthermore, C domain seems to be able to bind with the Gag-encoded capsid (CA) of HIV-1 and may affect the viral replication of HIV-1. A key residue of the active site is changed from Ala-103-CypA to Ser-239-hCyP33, which may affect the PPIase domain/substrates interactions.

Insulin and insulin-like growth factor 1 (IGF-1) share a homologous sequence, a similar three-dimensional structure and weakly overlapping biological activity, but IGF-1 folds into two thermodynamically stable disulfide isomers, while insulin folds into one unique stable tertiary structure. This is a very interesting phenomenon in which one amino acid sequence encodes two three-dimensional structures, and its molecular mechanism has remained unclear for a long time. In this study, the crystal structure of mini-IGF-1(2), a disulfide isomer of an artificial analog of IGF-1, was solved by the SAD/SIRAS method using our in-house X-ray source. Evidence was found in the structure showing that the intra-A-chain/domain disulfide bond of some molecules was broken; thus, it was proposed that disulfide isomerization begins with the breakdown of this disulfide bond. Furthermore, based on the structural comparison of IGF-1 and insulin, a new assumption was made that in insulin the several hydrogen bonds formed between the N-terminal region of the B-chain and the intra-A-chain disulfide region of the A-chain are the main reason for the stability of the intra-A-chain disulfide bond and for the prevention of disulfide isomerization, while Phe B1 and His B5 are very important for the formation of these hydrogen bonds. Moreover, the receptor binding property of IGF-1 was analyzed in detail based on the structural comparison of mini-IGF-1(2), native IGF-1, and small mini-IGF-1.

Renal tubular citrate transport is accomplished by electrogenic Na(+) coupled dicarboxylate transporter NaDC-1, a carrier subjected to regulation by acidosis. Trafficking of the Na(+)/H(+) exchanger NHE3 is controlled by NHE regulating factors NHERF-1 and NHERF-2 and the serum and glucocorticoid inducible kinase SGK1. To test for a possible involvement in NaDC-1 regulation, mRNA encoding NaDC-1 was injected into Xenopus oocytes with or without cRNA encoding NHERF-1, NHERF-2, SGK1, SGK2, SGK3, and/or the constitutively active form of the related protein kinase B ((T308,S473D)PKB). Succinate induced inward currents (I(succ)) were taken as a measure of transport rate. Coexpression of neither NHERF-1 nor NHERF-2 in NaDC-1 expressing oocytes significantly altered I(succ). On the other hand, coexpression of SGK1, SGK3, and (T308,S473D)PKB stimulated I(succ), an effect further stimulated by additional coexpression of NHERF-2 but not of NHERF-1. The action of the kinases and NHERF-2 may link urinary citrate excretion to proximal tubular H(+) secretion.

Mitogen-activated protein kinase (MAPK) cascade(s) is important for plant defense/stress responses. Though MAPKs have been identified and characterized in rice (Oryza sativa L.), a monocot cereal crop research model, the first upstream component of the kinase cascade, namely MAPK kinase kinase (MAPKKK) has not yet been identified. Here we report the cloning of a novel rice gene encoding a MAPKKK, OsEDR1, designated based on its homology with the Arabidopsis MAPKKK, AtEDR1. OsEDR1, a single copy gene in the genome of rice, encodes a predicted protein with molecular mass of 113046.13 and a pI of 9.03. Using our established two-week-old rice seedling in vitro model system, we show that OsEDR1 has a constitutive expression in seedling leaves and is further up-regulated within 15 min upon wounding by cut, treatment with the global signals jasmonic acid (JA), salicylic acid (SA), ethylene (ethephon, ET), abscisic acid, and hydrogen peroxide. In addition, protein phosphatase inhibitors, fungal elicitor chitosan, drought, high salt and sugar, and heavy metals also dramatically induce its expression. Moreover, OsEDR1 expression was altered by co-application of JA, SA, and ET, and required de novo synthesized protein factor(s) in its transient regulation. Furthermore, using an in vivo system we also show that OsEDR1 responds to changes in temperature and environmental pollutants-ozone and sulfur dioxide. Finally, OsEDR1 expression varied significantly in vegetative and reproductive tissues. These results suggest a role for OsEDR1 in defense/stress signalling pathways and development.

We reported previously that mouse ING1 homolog (mINGh), localized in the nucleus, enhanced cell death in HC11 mouse mammary epithelial cells. Analysis of the mINGh amino acid sequences revealed the presence of potential nuclear localization signal (NLS) and plant homeodomain (PHD) finger DNA binding domain. In the present study, NLS site in mINGh was determined using different pieces of mutant mINGh proteins, which were fused to green fluorescent protein (GFP), and transfected into HC11 cells. NLS of mINGh was split into two parts consisting of amino acids KEKK and KKLK. Mutation in NLS sites of mINGh resulted in no enhancement of the cell death when over-expressed. These results indicated that mINGh contains NLS of bipartite type, which is essential for the regulation of cell death.

We have isolated cDNA fragments that were originated from P450 1A1 in human brain by polymerase chain reaction (PCR) analysis. Their size matched up to what we expected based on the reported P450 1A1 mRNA sequence of human breast carcinoma cell line MCF-7. To confirm proper PCR products, they were subcloned and their nucleotide sequences were determined. This result showed that PCR products were actually derived from P450 1A1 mRNA in human brain. We demonstrated for the first time that human brain contains a P450 1A1-dependent metabolism system which seems to be important for understanding the metabolism of endogenous and exogenous compounds.

We have isolated cDNA fragments that were originated from P450 2E1 in rat brain by PCR analysis. Their size matched up to what we expected based on the reported sequence of rat liver P450 2E1 mRNA. Internal structure of the longest first-round PCR products were investigated by Southern blot analysis and "nested" PCR. Their results confirmed that PCR products actually originated from P450 2E1 mRNA in rat brain. RT-PCR was also carried out using P450 2E1 specific primers and the size of the product was exactly as we expected for P450 2E1. These experimental evidences should clarify the presence of P450 2E1 in rat brain.

The effect of myristicin on the expression of liver cytochrome P450s and its mRNA levels was examined in rats. Treatment of rats with myristicin (i.p., 500 mumol/kg) caused 2-20 fold increases in liver P450 1A1/2, 2B1/2, and 2E1 enzyme activities relative to controls. Immunoblot analysis using anti-rat liver P450 1A, 2B, and 2E1 showed that the increases in each of P450 protein levels by myristicin were consistent with those in enzyme activity levels. When increased levels of P450 mRNA by myristicin were examined by Northern blot analysis, levels of mRNA of P450 1A1/2 and 2B1/2 also increased. However, the level of P4502E1 mRNA did not increase. The total amount of spectrally detectable P450, also increased about 1.5-fold following treatment of myristicin. These results demonstrate that myristicin is an inducer of rat liver P450 1A1/2, 2B1/2, and 2E1, and that the induction involves increases in mRNA levels except in the case of P4502E1.

Induction of liver cytochrome P450 2B1 by beta-ionone was investigated in male and female Sprague Dawley rats. Administration of beta-ionone subcutaneously 72 and 48 hr before sacrificing the animals not only significantly induced the liver microsomal activity of pentoxyresorufin O-dealkylase, but also clearly increased in the level of cytochrome P450 2B1 protein. The induction of cytochrome P450 2B1 by beta-ionone was much greater in male rats than in female rats. A slot blot analysis showed that the mRNA level was increased from 6 hr after treatment with beta-ionone in male rats and from 12 hr after treatment in female rats. Taken together, the present results indicate for the first time that the induction of cytochrome P450 2B1 by beta-ionone might be regulated by the accumulation of mRNAs.

Cultured mouse hepatoma cell line Hepa-1c1c7 cells were treated with methoxsalen to assess the role of methoxsalen in the process of Cyp1a-1 induction. Treatment of Hepa-1c1c7 cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cyp1a-1, as indicated by analysis of 7-ethoxyresorufin O-deethylation (EROD) activity and P4501A1 protein. When methoxsalen and TCDD were both added to cultures, TCDD-inducible EROD activity was greatly suppressed by methoxsalen in a dose-dependent manner. We find that treatment of Hepa-1c1c7 cells with methoxsalen inhibited CYP1A1 mRNA induction by TCDD as well as the concomitant increase P4501A1 protein. Formation of DNA-protein complexes between the dioxin receptor and its DRE target was inhibited by methoxsalen, as determined by gel mobility shift assays using oligonucleotides corresponding to DRE 3 of the Cyp1a-1 gene. These results suggest that the inhibitory action of methoxsalen on TCDD induction of the Cyp1a-1 gene expression in Hepa-1c1c7 cells might be antagonism of the DNA binding potential of nuclear dioxin receptor.

A thiol-specific antioxidant protein (Protector Protein, PRP) was purified from human red blood cells (RBC). The PRP exists as a predominant protein in human RBC, which showed distinct thiol-specific antioxidant activities in the presence of dithiothreitol (DTT) as a reducing equivalent. The human RBC PRP (HRPRP) completely inhibited visible absorption spectral changes of oxyhemoglobin, DNA cleavage, and the peroxidation of RBC membrane by a nonenzymatic Fe3+/O2/thiol mixed-function oxidation system capable of generating hydroxyl radical. These observations suggest that HRPRP could act as a new type of antioxidant protein to maintain the RBC integrity by scavenging reactive oxygen species.

A family of Na+/H+ exchanger isoforms (called NHE1, NHE2, and NHE3) which exhibits a wide range of amiloride sensitivity has recently been cloned and characterized. A part of the domain, which determines amiloride sensitivity in the epithelial Na+/H+ exchanger isoform, NHE2, was identified by site-directed mutagenesis and functional studies using cDNAs stably expressed in a fibroblast cell line. It has previously been reported that AR300, an amiloride resistant mutant of the ubiquitous Na+/H+ exchanger isoform, NHE1, is 30-fold more resistant to methylpropyl amiloride (MPA) compared to NHE1 and contains a single amino acid substitution of L167F in the fourth putative transmembrane helix, which corresponds to L143 in NHE2. Therefore, in the present study point mutational substitutions were introduced into the equivalent of this fourth transmembrane helix of rabbit NHE2 (including Y144F; L143F; L143F and Y144F) to mimic the corresponding amino acids in NHE1, NHE3 (another epithelial isoform) and AR300, respectively. NHE2/L143F (mimicking NHE3) increased the IC50 for amiloride by 5-fold and for ethylisopropyl amiloride (EIPA) by 20-fold. Similarly, NHE2/L143F and Y144F (mimicking AR300) increased the resistance to both amiloride and EIPA by 10-fold. On the other hand, NHE2/Y144F (mimicking NHE1) did not affect the sensitivity to amiloride or EIPA, and this mutant, like wild type NHE2, is partially resistant to EIPA. Thus, amino acid 143 of NHE2 is critical for, but is not the only amino acid responsible for, amiloride and EIPA inhibition of Na+/H+ exchange. That none of the mutations studied altered the Na+ affinity of these Na+/H+ exchangers further suggests that amiloride binding and Na+ transport sites are not identical.