Iron uptake mediated by the plant-derived chelator nicotianamine in the small intestine.

Iron is an essential metal for all living organisms that is absorbed in the intestinal cells as a heme-chelated or free form. It is unclear how important plant-derived chelators, such as nicotianamine (NA), an organic small molecule that is ubiquitous in crops, vegetables, and various other foods, contribute to iron bioavailability in mammals. We performed electrophysiological assays with Xenopus laevis oocytes and radioactive tracer experiments with Caco-2 cells. The findings revealed that the proton-coupled amino acid transporter SLC36A1 (PAT1) transports iron in the form of NA-Fe (II) complex in vitro. Decreased expression of hPAT1 by RNA interference in Caco-2 cells reduced the uptake of NA-59Fe (II) complex. The uptake of inorganic 59Fe (II) was relatively unaffected. These results imply that PAT1 transports iron as a NA-Fe (II) complex. The rate of 59Fe absorption in the spleen, liver, and kidney was higher when mice were orally administered NA-59Fe (II) compared with free 59Fe (II). The profile of site-specific PAT1 expression in the mouse intestine coincided with those of NA and iron contents, which were the highest in the proximal jejunum. Orally administered NA-59Fe (II) complex in mice was detected in the proximal jejunum by thin layer chromatography. In contrast, much less 59Fe (or NA) was detected in the duodenum, where the divalent metal transporter SLC11A2 (DMT1) absorbs free Fe (II). The collective results revealed the role of PAT1 in NA-Fe (II) absorption in the intestine and potential implication of NA in iron uptake in mammals.

Generation and characterization of severe combined immunodeficiency rats.

Severe combined immunodeficiency (SCID) mice, the most widely used animal model of DNA-PKcs (Prkdc) deficiency, have contributed enormously to our understanding of immunodeficiency, lymphocyte development, and DNA-repair mechanisms, and they are ideal hosts for allogeneic and xenogeneic tissue transplantation. Here, we use zinc-finger nucleases to generate rats that lack either the Prkdc gene (SCID) or the Prkdc and Il2rg genes (referred to as F344-scid gamma [FSG] rats). SCID rats show several phenotypic differences from SCID mice, including growth retardation, premature senescence, and a more severe immunodeficiency without "leaky" phenotypes. Double-knockout FSG rats show an even more immunocompromised phenotype, such as the abolishment of natural killer cells. Finally, xenotransplantation of human induced pluripotent stem cells, ovarian cancer cells, and hepatocytes shows that SCID and FSG rats can act as hosts for xenogeneic tissue grafts and stem cell transplantation and may be useful for preclinical testing of new drugs.

Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.

Dysregulation of IFN system can lead to poor response to pegylated interferon and ribavirin therapy in chronic hepatitis C.

BACKGROUND: Despite being expensive, the standard combination of pegylated interferon (Peg-IFN)-α and ribavirin used to treat chronic hepatitis C (CH) results in a moderate clearance rate and a plethora of side effects. This makes it necessary to predict patient outcome so as to improve the accuracy of treatment. Although the antiviral mechanism of genetically altered IL28B is unknown, IL28B polymorphism is considered a good predictor of IFN combination treatment outcome. METHODOLOGY: Using microarray, we quantified the expression profile of 237 IFN related genes in 87 CH liver biopsy specimens to clarify the relationship between IFN pathway and viral elimination, and to predict patients' clinical outcome. In 72 out of 87 patients we also analyzed IL28B polymorphism (rs8099917). PRINCIPAL FINDINGS: Five IFN related-genes (IFI27, IFI 44, ISG15, MX1, and OAS1) had expression levels significantly higher in nonresponders (NR) than in normal liver (NL) and sustained virological responders (SVR); this high expression was also frequently seen in cases with the minor (TG or GG) IL28B genotype. The expression pattern of 31 IFN related-genes also differed significantly between NR and NL. We predicted drug response in NR with 86.1% accuracy by diagonal linear discriminant analysis (DLDA). CONCLUSION: IFN system dysregulation before treatment was associated with poor IFN therapy response. Determining IFN related-gene expression pattern based on patients' response to combination therapy, allowed us to predict drug response with high accuracy. This method can be applied to establishing novel antiviral therapies and strategies for patients using a more individual approach.

Virus-infection or 5'ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1.

In virus-infected cells, RIG-I-like receptor (RLR) recognizes cytoplasmic viral RNA and triggers innate immune responses including production of type I and III interferon (IFN) and the subsequent expression of IFN-inducible genes. Interferon-beta promoter stimulator 1 (IPS-1, also known as MAVS, VISA and Cardif) is a downstream molecule of RLR and is expressed on the outer membrane of mitochondria. While it is known that the location of IPS-1 is essential to its function, its underlying mechanism is unknown. Our aim in this study was to delineate the function of mitochondria so as to identify more precisely its role in innate immunity. In doing so we discovered that viral infection as well as transfection with 5'ppp-RNA resulted in the redistribution of IPS-1 to form speckle-like aggregates in cells. We further found that Mitofusin 1 (MFN1), a key regulator of mitochondrial fusion and a protein associated with IPS-1 on the outer membrane of mitochondria, positively regulates RLR-mediated innate antiviral responses. Conversely, specific knockdown of MFN1 abrogates both the virus-induced redistribution of IPS-1 and IFN production. Our study suggests that mitochondria participate in the segregation of IPS-1 through their fusion processes.