Increased expression of CD38 on endothelial cells in SARS-CoV-2 infection in cynomolgus macaques.

SARS-CoV-2 infection causes activation of endothelial cells (ECs), leading to dysmorphology and dysfunction. To study the pathogenesis of endotheliopathy, the activation of ECs in lungs of cynomolgus macaques after SARS-CoV-2 infection and changes in nicotinamide adenine dinucleotide (NAD) metabolism in ECs were investigated, with a focus on the CD38 molecule, which degrades NAD in inflammatory responses after SARS-CoV-2 infection. Activation of ECs was seen from day 3 after SARS-CoV-2 infection in macaques, with increases of intravascular fibrin and NAD metabolism-associated enzymes including CD38. In vitro, upregulation of CD38 mRNA in human ECs was detected after interleukin 6 (IL-6) trans-signaling induction, which was increased in the infection. In the presence of IL-6 trans-signaling stimulation, however, CD38 mRNA silencing induced significant IL-6 mRNA upregulation in ECs and promoted EC apoptosis after stimulation. These results suggest that upregulation of CD38 in patients with COVID-19 has a protective role against IL-6 trans-signaling stimulation induced by SARS-CoV-2 infection.

Suppression of viral RNA polymerase activity is necessary for persistent infection during the transformation of measles virus into SSPE virus.

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by measles virus (MV), which typically develops 7 to 10 years after acute measles. During the incubation period, MV establishes a persistent infection in the brain and accumulates mutations that generate neuropathogenic SSPE virus. The neuropathogenicity is closely associated with enhanced propagation mediated by cell-to-cell fusion in the brain, which is principally regulated by hyperfusogenic mutations of the viral F protein. The molecular mechanisms underlying establishment and maintenance of persistent infection are unclear because it is impractical to isolate viruses before the appearance of clinical signs. In this study, we found that the L and P proteins, components of viral RNA-dependent RNA polymerase (RdRp), of an SSPE virus Kobe-1 strain did not promote but rather attenuated viral neuropathogenicity. Viral RdRp activity corresponded to F protein expression; the suppression of RdRp activity in the Kobe-1 strain because of mutations in the L and P proteins led to restriction of the F protein level, thereby reducing cell-to-cell fusion mediated propagation in neuronal cells and decreasing neuropathogenicity. Therefore, the L and P proteins of Kobe-1 did not contribute to progression of SSPE. Three mutations in the L protein strongly suppressed RdRp activity. Recombinant MV harboring the three mutations limited viral spread in neuronal cells while preventing the release of infectious progeny particles; these changes could support persistent infection by enabling host immune escape and preventing host cell lysis. Therefore, the suppression of RdRp activity is necessary for the persistent infection of the parental MV on the way to transform into Kobe-1 SSPE virus. Because mutations in the genome of an SSPE virus reflect the process of SSPE development, mutation analysis will provide insight into the mechanisms underlying persistent infection.

Human metapneumovirus M2-2 protein inhibits RIG-I signaling by preventing TRIM25-mediated RIG-I ubiquitination.

Retinoic acid-inducible gene I (RIG-I) is a receptor that senses viral RNA and interacts with mitochondrial antiviral signaling (MAVS) protein, leading to the production of type I interferons and inflammatory cytokines to establish an antiviral state. This signaling axis is initiated by the K63-linked RIG-I ubiquitination, mediated by E3 ubiquitin ligases such as TRIM25. However, many viruses, including several members of the family Paramyxoviridae and human respiratory syncytial virus (HRSV), a member of the family Pneumoviridae, escape the immune system by targeting RIG-I/TRIM25 signaling. In this study, we screened human metapneumovirus (HMPV) open reading frames (ORFs) for their ability to block RIG-I signaling reconstituted in HEK293T cells by transfection with TRIM25 and RIG-I CARD (an N-terminal CARD domain that is constitutively active in RIG-I signaling). HMPV M2-2 was the most potent inhibitor of RIG-I/TRIM25-mediated interferon (IFN)-ß activation. M2-2 silencing induced the activation of transcription factors (IRF and NF-kB) downstream of RIG-I signaling in A549 cells. Moreover, M2-2 inhibited RIG-I ubiquitination and CARD-dependent interactions with MAVS. Immunoprecipitation revealed that M2-2 forms a stable complex with RIG-I CARD/TRIM25 via direct interaction with the SPRY domain of TRIM25. Similarly, HRSV NS1 also formed a stable complex with RIG-I CARD/TRIM25 and inhibited RIG-I ubiquitination. Notably, the inhibitory actions of HMPV M2-2 and HRSV NS1 are similar to those of V proteins of several members of the Paramyxoviridae family. In this study, we have identified a novel mechanism of immune escape by HMPV, similar to that of Pneumoviridae and Paramyxoviridae family members.

Upregulation of viral RNA polymerase activity promotes adaptation of SSPE virus to neuronal cells.

Subacute sclerosing panencephalitis (SSPE) is a rare progressive neurodegenerative disease caused by measles virus variants (SSPE viruses) that results in eventual death. Amino acid substitution(s) in the viral fusion (F) protein are key for viral propagation in the brain in a cell-to-cell manner, a specific trait of SSPE viruses, leading to neuropathogenicity. In this study, we passaged an SSPE virus in cultured human neuronal cells and isolated an adapted virus that propagated more efficiently in neuronal cells and exhibited increased cell-to-cell fusion. Contrary to our expectation, the virus harbored mutations in the large protein, a viral RNA-dependent RNA polymerase, and in the phosphoprotein, its co-factor, rather than in the F protein. Our results imply that upregulated RNA polymerase activity, which increases F protein expression and cell-to-cell fusion, could be a viral factor that provides a growth advantage and contributes to the adaptation of SSPE viruses to neuronal cells.

M protein of subacute sclerosing panencephalitis virus, synergistically with the F protein, plays a crucial role in viral neuropathogenicity.

Subacute sclerosing panencephalitis (SSPE) is a rare fatal neurodegenerative disease caused by a measles virus (MV) variant, SSPE virus, that accumulates mutations during long-term persistent infection of the central nervous system (CNS). Clusters of mutations identified around the matrix (M) protein in many SSPE viruses suppress productive infectious particle release and accelerate cell-cell fusion, which are features of SSPE viruses. It was reported, however, that these defects of M protein function might not be correlated directly with promotion of neurovirulence, although they might enable establishment of persistent infection. Neuropathogenicity is closely related to the character of the viral fusion (F) protein, and amino acid substitution(s) in the F protein of some SSPE viruses confers F protein hyperfusogenicity, facilitating viral propagation in the CNS through cell-cell fusion and leading to neurovirulence. The F protein of an SSPE virus Kobe-1 strain, however, displayed only moderately enhanced fusion activity and required additional mutations in the M protein for neuropathogenicity in mice. We demonstrated here the mechanism for the M protein of the Kobe-1 strain supporting the fusion activity of the F protein and cooperatively inducing neurovirulence, even though each protein, independently, has no effect on virulence. The occurrence of SSPE has been estimated recently as one in several thousand in children who acquired measles under the age of 5 years, markedly higher than reported previously. The probability of a specific mutation (or mutations) occurring in the F protein conferring hyperfusogenicity and neuropathogenicity might not be sufficient to explain the high frequency of SSPE. The induction of neurovirulence by M protein synergistically with moderately fusogenic F protein could account for the high frequency of SSPE.

Annexin A1 accounts for an anti-inflammatory binding target of sesamin metabolites.

Sesamin [(7α,7'α,8α,8'α)-3,4:3',4'-bis(methylenedioxy)-7,9':7',9-diepoxylignane] is a major lignan in sesame seeds. Sesamin is converted to the catechol metabolite, SC1 [(7α,7'α,8α,8'α)-3',4'-methylenedioxy-7,9':7',9-diepoxylignane-3,4-diol] with anti-inflammatory effects after oral administration. However, its molecular target remains unknown. Analysis using high-performance affinity nanobeads led to the identification of annexin A1 (ANX A1) as an SC1-binding protein. SC1 was found to bind to the annexin repeat 3 region of ANX A1 with a high-affinity constant (Kd = 2.77 µmol L-1). In U937 cells, SC1 exhibited an anti-inflammatory effect dependent on ANX A1. Furthermore, administration of sesamin or SC1 attenuated carbon tetrachloride-induced liver damage in mice and concurrently suppressed inflammatory responses dependent on ANX A1. The mechanism involved SC1-induced ANX A1 phosphorylation at serine 27 that facilitates extracellular ANX A1 release. Consequently, the ANX A1 released into the extracellular space suppressed the production of tumor necrosis factor α. This study demonstrates that ANX A1 acts as a pivotal target of sesamin metabolites to attenuate inflammatory responses.

Multiple-endpoint genotoxicity assay for colon carcinogen 1,2-dimethylhydrazine.

Human risk assessment of the toxic potency of chemicals typically includes genotoxicity assays for predicting carcinogenicity. Gene mutation frequency and chromosomal aberration are two major genotoxicity endpoints in standardized in vitro and in vivo assays. The weight-of-evidence approach in risk assessment is more focused on in vivo assay results; however, animal welfare considerations are aimed at the reduction, replacement, and refinement (3R's) of animal experiments, including a reduction in the number of experimental animals. Proposals to reduce experimental animals in genotoxicity testing include the incorporation of genotoxicity endpoint(s) into other toxicological studies and the combination of two or more assays detecting different genotoxicity endpoints in the same animals. In this study, we used 1,2-dimethylhydrazine as a model chemical of colon carcinogen to assess gene mutation frequency and chromosomal aberration in vivo simultaneously. Specifically, a gene mutation frequency assay was combined with a multiple-organ micronucleus test (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic rats. Both gpt mutant frequency and micronucleated cell frequency significantly increased in colon and liver but not in bone marrow. Interestingly, we found that the colon carcinogen induced both gene mutations and micronuclei in the targeted colon tissue. Thus, we demonstrated that the mechanism of a carcinogen could be derived from an animal experiment using a lower number of experimental animals as currently recommended. Moreover, a significant increase in mutant frequency in colon and liver was already observed on the first day after treatment completion, as well as on the third day, which is the guideline-recommended period. Thus, this endpoint is compatible with other genotoxicity assays. We confirmed that performing the micronucleus assay in combination with a gene mutation assay in F344 gpt delta transgenic rats is useful to evaluate different genotoxic endpoints simultaneously in the same animals, which reduces the number of experimental animals.

Target-specific micronucleus induction by colon carcinogens: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 1,2-dimethylhydrazine.

Genotoxicity occurring at the target organs of carcinogenesis is important for understanding the mechanisms of chemical carcinogenicity and also for setting of threshold estimation. In vivo gene mutations have been evaluated by transgenic animal models in which any organ can be targeted; however, the methodologies that have been applied to assess chromosomal aberrations including micronucleus induction, are organ restricted, (often to bone marrow hematopoietic cells, as a common example). For food and food-related chemicals, the digestive tract is the important target organ as it is the organ of first contact. In the present study, we used 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 1,2-dimethylhydrazine (DMH) as model chemicals of carcinogens primarily targeting the colon. We evaluated the applicability of colon cells and hepatocytes, together with bone marrow cells, in the micronucleus assay. Both model chemicals induced micronuclei in the colon, which is the target organ of these carcinogens, after short- and long-term treatment(s). The results demonstrate the target specificity of micronucleus induction and the assay using organs other than bone marrow will play an important role in understanding the mechanism of carcinogenicity and predicting new carcinogenic agents.

Oncogenic role of TYRO3 receptor tyrosine kinase in the progression of pancreatic cancer.

The expression and functions of TYRO3, a member of the TAM receptor tyrosine kinase family, in pancreatic cancer (PC) have not been specifically elucidated. In this study, we confirmed TYRO3 expression in five human PC cell lines (PANC-1, MIA PaCa-2, BxPC-3, AsPC-1, and PK-9) using Western blotting. TYRO3 silencing and overexpression studies have revealed that TYRO3 promotes cell proliferation and invasion in PC via phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK). Using a mouse xenograft model, we showed that tumor growth was significantly suppressed in mice subcutaneously inoculated with TYRO3-knockdown PC cells compared with mice inoculated with control PC cells. Furthermore, TYRO3 expression was examined in PC tissues obtained from 106 patients who underwent pancreatic resection for invasive ductal carcinoma through immunohistochemical staining. TYRO3-positive patients had poor prognoses for overall survival and disease-specific survival compared with TYRO3-negative patients. Multivariate analysis revealed that TYRO3 expression is an independent prognostic factor for overall survival. Our study demonstrates the critical role of TYRO3 in PC progression through Akt and ERK activation and suggests TYRO3 as a novel promising target for therapeutic strategies against PC.

System xc- in microglia is a novel therapeutic target for post-septic neurological and psychiatric illness.

Post-septic neurological and psychiatric illness (PSNPI) including dementia and depression may be observed after sepsis. However, the etiology of PSNPI and therapeutic treatment of PSNPI are unclear. We show that glutamate produced from microglia through the activity of system xc- plays a role in PSNPI. We established a mouse model of PSNPI by lipopolysaccharide (LPS) treatment that shows a disturbance of short/working memory and depression-like hypoactivity. Glutamate receptor antagonists (MK801 and DNQX) reduced these phenotypes, and isolated microglia from LPS-treated mice released abundant glutamate. We identified system xc- as a source of the extracellular glutamate. xCT, a component of system xc-, was induced and expressed in microglia after LPS treatment. In xCT knockout mice, PSNPI were decreased compared to those in wildtype mice. Moreover, TNF-α and IL-1ß expression in wildtype mice was increased after LPS treatment, but inhibited in xCT knockout mice. Thus, system xc- in microglia may be a therapeutic target for PSNPI. The administration of sulfasalazine, an inhibitor of xCT, in symptomatic and post-symptomatic mice improved PSNPI. Our results suggest that glutamate released from microglia through system xc- plays a critical role in the manifestations of PSNPI and that system xc- may be a therapeutic target for PSNPI.

Peptide barcoding for establishment of new types of genotype-phenotype linkages.

Measuring binding properties of binders (e.g., antibodies) is essential for developing useful experimental reagents, diagnostics, and pharmaceuticals. Display technologies can evaluate a large number of binders in a high-throughput manner, but the immobilization effect and the avidity effect prohibit the precise evaluation of binding properties. In this paper, we propose a novel methodology, peptide barcoding, to quantitatively measure the binding properties of multiple binders without immobilization. In the experimental scheme, unique peptide barcodes are fused with each binder, and they represent genotype information. These peptide barcodes are designed to have high detectability for mass spectrometry, leading to low identification bias and a high identification rate. A mixture of different peptide-barcoded nanobodies is reacted with antigen-coated magnetic beads in one pot. Peptide barcodes of functional nanobodies are cleaved on beads by a specific protease, and identified by selected reaction monitoring using triple quadrupole mass spectrometry. To demonstrate proof-of-principle for peptide barcoding, we generated peptide-barcoded anti-CD4 nanobody and anti-GFP nanobody, and determined whether we could simultaneously quantify their binding activities. We showed that peptide barcoding did not affect the properties of the nanobodies, and succeeded in measuring the binding activities of these nanobodies in one shot. The results demonstrate the advantages of peptide barcoding, new types of genotype-phenotype linkages.

Integration of micronucleus tests with a gene mutation assay in F344 gpt delta transgenic rats using benzo[a]pyrene.

Reduction of the number of animals used in in vivo genotoxicity tests is encouraged. For this purpose, we conducted integrated toxicity tests combining gene mutation assays with multiple-organ micronucleus (MN) tests (peripheral blood, bone marrow, liver, and colon) in F344 gpt delta transgenic (Tg) rats. Seven-week-old male F344 gpt delta rats were orally administered 62.5 or 125 mg/kg/day benzo[a]pyrene (B[a]P) for 28 days. One day after the final day of treatment (day 29) and three days after the final treatment (day 31), bone marrow, liver, and colon samples were collected, and mutation assays and MN tests were performed. The gpt mutant frequency (MF) significantly increased in bone marrow, liver and colon but MN induction was only significant in bone marrow but not in liver and colon. Similarly MN induction was only observed in bone marrow in non-Tg F344 rats. In peripheral blood obtained on day 4, 15, 29, 31, a time-dependent increase was observed in reticulocyte MN frequency during the treatment. Thus, our integrated method successfully detected both gene mutations and MN induction caused by B[a]P. In addition, no significant differences were observed between sampling times (day 29 versus 31), suggesting that sampling on day 29 is also valid to evaluate gene mutations. On the other hand, MN results in bone marrow and peripheral blood were different depending on the sampling day. An appropriate sampling day should be designated according to which assays are integrated. We confirmed that integration of the MN test with a gene mutation assay using F344 gpt delta Tg rats is useful to evaluate different endpoints related to genotoxicity using the same animals and to reduce animal use.

Sendai Virus V Protein Inhibits the Secretion of Interleukin-1ß by Preventing NLRP3 Inflammasome Assembly.

Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1ß (IL-1ß) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1ß maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus [SeV V(-)] induced markedly greater amounts of IL-1ß than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1ß secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1ß activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly was observed. Therefore, SeV V protein likely inhibits NLRP3 self-oligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1ß secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1ß activation.IMPORTANCE The present study demonstrates that the V protein of SeV, Nipah virus, and human parainfluenza virus type 2 interacts with NLRP3 to inhibit NLRP3 inflammasome activation, potentially suggesting a novel strategy by which viruses evade the host innate immune response. As all members of the Paramyxovirinae subfamily carry similar V genes, this new finding may also lead to identification of novel therapeutic targets for paramyxovirus infection and related diseases.

α-Tocopherol promotes HaCaT keratinocyte wound repair through the regulation of polarity proteins leading to the polarized cell migration.

In many developed countries including Japan, how to care the bedridden elderly people with chronic wounds such as decubitus becomes one of the most concerned issues. Although antioxidant micronutrients including vitamin E, especially α-tocopherol (α-Toc), are reported to shorten a period of wound closure, the promoting effect of α-Toc on wound healing independent of its antioxidant activity remains to be fully elucidated. The aim of this study was to examine whether α-Toc affects wound-mediated HaCaT keratinocyte polarization process including the recruitment of polarity regulating proteins, leading to wound repair independently of its antioxidant activity. We investigated the effects of α-Toc and other antioxidants such as Trolox, a cell-permeable α-Toc analog on the migration, proliferation, and cell polarization of HaCaT keratinocytes after wounding. We analyzed the localization and complex formation of polarity proteins, partitioning defective 3 (Par3), and atypical protein kinase C (aPKC), and aPKC activity by immunohistochemistry, immunoprecipitation analyses, and in vitro kinase assays, respectively. α-Toc but not other antioxidants enhanced the wound closure and cell polarization in HaCaT keratinocytes after wounding. α-Toc regulated the localization and complex formation of Par3 and aPKC during wound healing. Knockdown of aPKC or Par3 abrogated α-Toc-mediated promotion of the wound closure and cell polarization in HaCaT keratinocytes. Furthermore, aPKC kinase activity was significantly increased in α-Toc-treated cells through activation of phosphatidylinositol 3-kinase/Akt signaling pathway. These results suggest that α-Toc promotes HaCaT keratinocyte wound repair by regulating the aPKC kinase activity and the formation of aPKC-Par3 complex. © 2017 BioFactors, 44(2):180-191, 2018.

Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells.

Human metapneumovirus (HMPV) has the ability to inhibit Toll-like receptor 7 (TLR7)- and TLR9-dependent alpha interferon (IFN-α) production by plasmacytoid dendritic cells (pDCs). However, the inhibition mechanism remains largely unknown. To identify viral proteins responsible for this inhibition, we performed a screening of HMPV open reading frames (ORFs) for the ability to block TLR7/9-dependent signaling reconstituted in HEK293T cells by transfection with myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), IKKα, and IFN regulatory factor 7 (IRF7). This screening demonstrated that the M2-2 protein was the most potent inhibitor of TLR7/9-dependent IFN-α induction. A recombinant HMPV in which the M2-2 ORF was silenced indeed induced greater IFN-α production by human pDCs than wild-type HMPV did. Immunoprecipitation experiments showed direct physical association of the M2-2 protein with the inhibitory domain (ID) of IRF7. As a natural consequence of this, transfection of IRF7 lacking the ID, a constitutively active mutant, resulted in activation of the IFN-α promoter even in the presence of M2-2. Bioluminescence resonance energy transfer assays and split Renilla luciferase complementation assays revealed that M2-2 inhibited MyD88/TRAF6/IKKα-induced homodimerization of IRF7. In contrast, expression of the M2-2 protein did not result in inhibition of IPS-1-induced homodimerization and resultant activation of IRF7. This indicates that inhibition of MyD88/TRAF6/IKKα-induced IRF7 homodimerization does not result from a steric effect of M2-2 binding. Instead, it was found that M2-2 inhibited MyD88/TRAF6/IKKα-induced phosphorylation of IRF7 on Ser477. These results suggest that M2-2 blocks TLR7/9-dependent IFN-α induction by preventing IRF7 homodimerization, possibly through its effects on the phosphorylation status of IRF7.IMPORTANCE The family Paramyxoviridae is divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae Members of the subfamily Paramyxovirinae have the ability to inhibit TLR7/9-dependent IFN-α production, and the underlying inhibition mechanism has been intensively studied. In contrast, little is known about how members of the subfamily Pneumovirinae regulate IFN-α production by pDCs. We identified the M2-2 protein of HMPV, a member of the subfamily Pneumovirinae, as a negative regulator of IFN-α production by pDCs and uncovered the underlying mechanism. This study explains in part why the M2-2 knockout recombinant HMPV is attenuated and further suggests that M2-2 is a potential target for HMPV therapy.

Mechanisms of chromosomal aberrations induced by sesamin metabolites in Chinese hamster lung cells.

Sesamin is a major lignan in sesame seeds and oil. We previously demonstrated that sesamin induces chromosomal aberrations (CA) in Chinese hamster lung (CHL/IU) cells in the presence of a metabolic activation system (S9 mix), although no genotoxicity was detected in vivo. To clarify the mechanism of CA induction by sesamin, we identified its principal active metabolite. A mono-catechol derivative, [2-(3,4-methylenedioxyphenyl)-6-(3,4-dihydroxyphenyl)-3,7-dioxabi-cyclo[3.3.0]octane (SC-1)], was previously identified in culture medium when sesamin was incubated with S9 mix. In the present study, we show that SC-1 induces CA in CHL/IU cells but not in human hepatoblastoma (HepG2) cells. SC-1 was unstable in culture medium. Addition of glutathione (GSH) to the incubation mixture decreased the rate of decomposition and also suppressed induction of CA in CHL/IU cells. These results indicate that SC-1 itself may not contribute to the induction of CA. Two GSH adducts of SC-1 were identified when SC-1 was incubated with GSH, suggesting that SC-1 was converted to the semiquinone/quinone form and then conjugated with GSH in the culture medium. Sodium sulfite (a quinone-responsive compound) also suppressed CA induction by SC-1. These findings strongly suggest that SC-1 is oxidized to semiquinone/quinone derivatives extracellularly in culture medium, that these derivatives are responsible for the induction of CA in CHL/IU cells, and therefore that the positive results obtained with sesamin in in vitro CA tests using CHL/IU cells may not be relevant to the assessment of in vivo activity.

Changes in coumarin kinetics and subcellular localization of CYP2E1 contribute to bile duct damage and reduce hepatocellular damage after repeated administration of coumarin in rats.

Coumarin exhibits different hepatotoxicity in rats depending on the administration frequency. To investigate the underlying mechanisms for the differences, we administered coumarin to rats as a single dose or repeatedly for 4 weeks. We found large increases in blood levels of liver enzymes and noticeable centrilobular hepatic necrosis after a single dose of coumarin. After repeated administration, enzyme levels mildly increased, while those of γ-GTP and total bilirubin significantly increased, suggesting bile duct damage. In the control group, cytochrome P450 2E1 (CYP2E1) showed a diffuse subcellular distribution but accumulated within the hepatocyte endoplasmic reticulum after repeated coumarin administration. The maximum blood concentrations of coumarin and its metabolites were significantly lower upon repeated administration. The results suggest that changes in coumarin pharmacokinetics and CYP2E1 subcellular distribution contribute to resistance to coumarin-induced hepatic necrosis, while cytotoxicity of metabolic conjugates shown in vitro may contribute to bile duct damage upon repeated coumarin administration.

Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway.

Necroptosis is a form of necrotic cell death that requires the activity of the death domain-containing kinase RIP1 and its family member RIP3. Necroptosis occurs when RIP1 is deubiquitinated to form a complex with RIP3 in cells deficient in the death receptor adapter molecule FADD or caspase-8. Necroptosis may play a role in host defense during viral infection as viruses like vaccinia can induce necroptosis while murine cytomegalovirus encodes a viral inhibitor of necroptosis. To see how general the interplay between viruses and necroptosis is, we surveyed seven different viruses. We found that two of the viruses tested, Sendai virus (SeV) and murine gammaherpesvirus-68 (MHV68), are capable of inducing dramatic necroptosis in the fibrosarcoma L929 cell line. We show that MHV68-induced cell death occurs through the cytosolic STING sensor pathway in a TNF-dependent manner. In contrast, SeV-induced death is mostly independent of TNF. Knockdown of the RNA sensing molecule RIG-I or the RIP1 deubiquitin protein, CYLD, but not STING, rescued cells from SeV-induced necroptosis. Accompanying necroptosis, we also find that wild type but not mutant SeV lacking the viral proteins Y1 and Y2 result in the non-ubiquitinated form of RIP1. Expression of Y1 or Y2 alone can suppress RIP1 ubiquitination but CYLD is dispensable for this process. Instead, we found that Y1 and Y2 can inhibit cIAP1-mediated RIP1 ubiquitination. Interestingly, we also found that SeV infection of B6 RIP3-/- mice results in increased inflammation in the lung and elevated SeV-specific T cells. Collectively, these data identify viruses and pathways that can trigger necroptosis and highlight the dynamic interplay between pathogen-recognition receptors and cell death induction.

A residue located at the junction of the head and stalk regions of measles virus fusion protein regulates membrane fusion by controlling conformational stability.

The fusion (F) protein of measles virus performs refolding from the thermodynamically metastable prefusion form to the highly stable postfusion form via an activated unstable intermediate stage, to induce membrane fusion. Some amino acids involved in the fusion regulation cluster in the heptad repeat B (HR-B) domain of the stalk region, among which substitution of residue 465 by various amino acids revealed that fusion activity correlates well with its side chain length from the Cα (P<0.01) and van der Waals volume (P<0.001), except for Phe, Tyr, Trp, Pro and His carrying ring structures. Directed towards the head region, longer side chains of the non-ring-type 465 residues penetrate more deeply into the head region and may disturb the hydrophobic interaction between the stalk and head regions and cause destabilization of the molecule by lowering the energy barrier for refolding, which conferred the F protein enhanced fusion activity. Contrarily, the side chain of ring-type 465 residues turned away from the head region, resulting in not only no contact with the head region but also extensive coverage of the HR-B surface, which may prevent the dissociation of the HR-B bundle for initiation of membrane fusion and suppress fusion activity. Located in the HR-B domain just at the junction between the head and stalk regions, amino acid 465 is endowed with a possible ability to either destabilize or stabilize the F protein depending on its molecular volume and the direction of the side chain, regulating fusion activity of measles virus F protein.

Evaluation of single-dose RBC Pig-a and PIGRET assays in detecting the mutagenicity of thiotepa in rats.

The Pig-a assay, which uses reticulocytes (PIGRET assay) as target cells, is anticipated to detect mutagenicity at earlier time points than the RBC Pig-a assay, which uses all red blood cells as target cells. As part of a collaborative study conducted by the Mammalian Mutagenicity Study (MMS) Group, we evaluated the PIGRET and RBC Pig-a assays to detect Pig-a gene mutations induced by the carcinogen thiotepa. A single dose of thiotepa at 7.5, 15, and 30mg/kg was administered to 8-week-old male Sprague-Dawley rats by oral gavage. PIGRET and RBC Pig-a assays were performed using peripheral blood collected from rats 7, 14, and 28days after thiotepa administration (Day 0 as the day of administration), and the resulting Pig-a mutant frequencies (MFs) were compared. Increased Pig-a MF was observed from Day 7 onwards using the PIGRET assay. Pig-a MF remained fairly constant thereafter until Day 28 in the 30mg/kg group, whereas it peaked on Day 14 in the 7.5 and 15mg/kg groups. Using the RBC Pig-a assay, on the other hand, no significant increase in MF was observed at any of the dosages on Days 7, 14, or 28. These findings show that Pig-a gene mutations following a single dose of thiotepa were detected using the PIGRET assay but not the RBC Pig-a assay, which suggests that PIGRET assay is more suitable than RBC Pig-a assay for evaluating the in vivo mutagenicity by a single dose.