Hiroshi Iwata

Role of TAK1 and TAB1 in BMP signaling in early Xenopus development.

Transforming growth factor‐β (TGF‐β) superfamily members elicit signals through stimulation of serine/threonine kinase receptors. Recent studies of this signaling pathway have identified two types of novel mediating molecules, the Smads and TGF‐β activated kinase 1 (TAK1). Smads were shown to mimic the effects of bone morphogenetic protein (BMP), activin and TGF‐β. TAK1 and TAB1 were identified as a MAPKKK and its activator, respectively, which might be involved in the up‐regulation of TGF‐β superfamily‐induced gene expression, but their biological role is poorly understood. Here, we have examined the role of TAK1 and TAB1 in the dorsoventral patterning of early Xenopus embryos. Ectopic expression of Xenopus TAK1 (xTAK1) in early embryos induced cell death. Interestingly, however, concomitant overexpression of bcl‐2 with the activated form of xTAK1 or both xTAK1 and xTAB1 in dorsal blastomeres not only rescued the cells but also caused the ventralization of the embryos. In addition, a kinase‐negative form of xTAK1 (xTAK1KN) which is known to inhibit endogenous signaling could partially rescue phenotypes generated by the expression of a constitutively active BMP‐2/4 type IA receptor (BMPR‐IA). Moreover, xTAK1KN could block the expression of ventral mesoderm marker genes induced by Smad1 or 5. These results thus suggest that xTAK1 and xTAB1 function in the BMP signal transduction pathway in Xenopus embryos in a cooperative manner.

High Catalytic Activity of Human Cytochrome P450 Co-expressed with Human NADPH-Cytochrome P450 Reductase in Escherichia coli

Forms of human cytochrome P450 (P450 or CYP), such as CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were expressed or co-expressed together with human NADPH-P450 reductase in Escherichia coli. When P450 was expressed alone in E. coli, the expression level of holo-P450 ranged from 310 to 1620 nmol/L of culture. The expression level of holo-P450 decreased by co-expression with the reductase, and the level ranged from 66 to 381 nmol/L of culture. The expression level of the reductase varied depending on the forms of P450 co-expressed, and ranged from 204 to 937 U/L of culture. We assayed the catalytic activity of P450 using E. coli cells disrupted by freeze-thaw. When co-expressed with the reductase, human P450 catalyzed the oxidation of representative substrates at efficient rates. The rates appeared comparable to the reported activities of P450 in a reconstituted system containing purified preparations of P450 and the reductase.

Inhibition of Virus Production in JC Virus-Infected Cells by Postinfection RNA Interference

ABSTRACT RNA interference has been applied for the prevention of virus infections in mammalian cells but has not succeeded in eliminating infections from already infected cells. We now show that the transfection of JC virus-infected SVG-A human glial cells with small interfering RNAs that target late viral proteins, including agnoprotein and VP1, results in a marked inhibition both of viral protein expression and of virus production. RNA interference directed against JC virus genes may thus provide a basis for the development of new strategies to control infections with this polyomavirus.

Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines

Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity. They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT). To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain. The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase. The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine compared with the parent Ames tester strain TA1538. The minimum concentrations of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively. When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO. These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.

Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection.

n Abstractn n Polyomavirus JC (JCV) infection causes the fatal human demyelinating disease, progressive multifocal leukoencephalopathy. Although the initial interaction of JCV with host cells occurs through direct binding of the major viral capsid protein (VP1) with cell-surface molecules possessing sialic acid, these molecules have not yet been identified. In order to isolate monoclonal antibodies which inhibit attachment of JCV, we established an immunoscreening system using virus-like particles consisting of the VP1. Using this system, among monoclonal antibodies against the cell membrane fraction from JCV-permissive human neuroblastoma IMR-32 cells, we isolated a monoclonal antibody designated as 24D2 that specifically inhibited attachment and infection of JCV to IMR-32 cells. The antibody 24D2 recognized a single molecule of around 60kDa in molecular weight in the IMR-32 membrane fraction. Immunohistochemical staining with 24D2 demonstrated immunoreactivity in the cell membrane of JCV-permissive cell lines and glial cells of the human brain. These results suggested that the molecule recognized by 24D2 plays a role in JCV infection, and that it might participate as a receptor or a co-receptor in JCV attachment and entry into the cells.n n