An efficient method oftransgene modulation in fertilized eggs has been developed that uses

Site-directed DNA integration has been achieved by using a pair of mutant lox sites, a right element (RE) mutant lox site and a left element (LE) mutant lox site [Albertet al. (1995)Plant J., 7, 649-659], in mouse embryonic stem (ES) cells. We established ES cell lines carrying a single copy of the wild-type lox Por LE mutant lox site as a target and examined the frequency of site-specific integration of a targeting vector carrying a loxP or RE mutant lox site induced by Cre transient expression. Since our targeting vector contains a complete neo gene, random integrants can form colonies as in the case of a gene targeting event through homologous recombination. With our system, the frequency of site-specific integration via the mutant lox sites reached a maximum of 16%. In contrast, the wild-type loxP sites yielded very low frequencies (<0.5%) of site-specific integration events. This mutatedloxsystem will be useful for 'knock-in' integration of DNA in ES cells.

The Beclin 1-Vps34 complex, the core component of the class III phosphatidylinositol-3 kinase (PI3K-III), binds Atg14L or UVRAG to control different steps of autophagy. However, the mechanism underlying the control of PI3K-III activity remains elusive. Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity. Through its MIT domain, NRBF2 binds Atg14L directly and enhances Atg14L-linked Vps34 kinase activity and autophagy induction. NRBF2 deficient cells exhibit enhanced vulnerability to ER stress that is reversed by re-introducing exogenous NRBF2. NRBF2 deficient mice develop focal liver necrosis and ductular reaction, accompanied by impaired Atg14L-linked Vps34 activity and autophagy, though the mice show no increased mortality. Our data reveals a key role for NRBF2 in the assembly of the specific Atg14L-Beclin 1-Vps34-Vps15 complex for autophagy induction. Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury.

SummaryThe selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the [3-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the o~1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with 0t(1,3/1,4) fucosyltransferasespecific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only (B16F10hygro). Normal mice injected with B16F10ft and B16FlOhygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.T umor metastasis is a multistep process requiring detachment of malignant cells from the primary tumor mass, penetration of blood and/or lymph vessels, evasion of immune surveillance, attachment to endothelium of distant organs, penetration of the secondary host tissue, and formation of new tumor colonies. The ability of tumor cells to selectively adhere to endothelium of certain organs may be responsible in part for preferential metastatic patterns associated with different tumor types. Interaction between tumor cells and host tissue endothelium is thought to be mediated by adhesion receptor-ligand pairs, some of which are involved in physiologic leukocyte-endothelial interactions (1). Tumor cells express various combinations of cell surface molecules that may serve as ligands for endothelial cell surface receptors (2-7), which are typically induced upon stimulation by mediators of inflammation (2-4). A local inflammatory response may therefore facilitate circulating tumor cell adhesion and arrest.Two members of the selectin family of adhesion molecules, E-and P-selectin, are transiently expressed on endothelial cells after stimulation by IL-1 and TNF-ot. Both re-L. Biancone and M. Araki contributed equally to this work. ceptors recognize sialylated fucosylated lactosaminoglycans on the surface of various leukocyte subsets and promote leukocyte rolling on the cytokine-activated endothelial surface, facilitating subsequent leukocyte arrest and extravasation to sites of injury (4). E-selectin has been proposed to facilitate attachment of tumor cells to endothelium in similar fashion (4). This sugg...

Tissue damage by oxidative stress is a key pathogenic mechanism in various diseases, including AKI and CKD. Thus, early detection of oxidative tissue damage is important. Using a tRNA-specific modified nucleoside 1-methyladenosine (m1A) antibody, we show that oxidative stress induces a direct conformational change in tRNA structure that promotes subsequent tRNA fragmentation and occurs much earlier than DNA damage. In various models of tissue damage (ischemic reperfusion, toxic injury, and irradiation), the levels of circulating tRNA derivatives increased rapidly. In humans, the levels of circulating tRNA derivatives also increased under conditions of acute renal ischemia, even before levels of other known tissue damage markers increased. Notably, the level of circulating free m1A correlated with mortality in the general population (n=1033) over a mean follow-up of 6.7 years. Compared with healthy controls, patients with CKD had higher levels of circulating free m1A, which were reduced by treatment with pitavastatin (2 mg/d; n=29). Therefore, tRNA damage reflects early oxidative stress damage, and detection of tRNA damage may be a useful tool for identifying organ damage and forming a clinical prognosis.