The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activationtagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and Os-NAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression. activation tagging ͉ bioavailability ͉ hemoglobin ͉ metal homeostasis

SummaryWe generated rice lines with increased content of nicotianamine (NA), a key ligand for metal transport and homeostasis. This was accomplished by activation tagging of rice nicotianamine synthase 2 (OsNAS2). Enhanced expression of the gene resulted in elevated NA levels, greater Zn accumulations and improved plant tolerance to a Zn deficiency. Expression of Zn-uptake genes and those for the biosynthesis of phytosiderophores (PS) were increased in transgenic plants. This suggests that the higher amount of NA led to greater exudation of PS from the roots, as well as stimulated Zn uptake, translocation and seed-loading. In the endosperm, the OsNAS2 activation-tagged line contained up to 20-fold more NA and 2.7-fold more zinc. Liquid chromatography combined with inductively coupled plasma mass spectrometry revealed that the total content of zinc complexed with NA and 2¢-deoxymugineic acid was increased 16-fold. Mice fed with OsNAS2-D1 seeds recovered more rapidly from a zinc deficiency than did control mice receiving WT seeds. These results demonstrate that the level of bio-available zinc in rice grains can be enhanced significantly by activation tagging of OsNAS2.

The heterogeneous cellular composition of the mammalian renal collecting duct enables regulation of fluid, electrolytes, and acid-base homeostasis, but the molecular mechanism of its development has yet to be elucidated. The Notch signaling pathway is involved in cell fate determination and has been implicated in proximal-distal patterning in the mammalian kidney. To investigate the role of Notch signaling in renal collecting duct development, we generated mice in which Mind bomb-1 (Mib1), an E3 ubiquitin ligase required for the initiation of Notch signaling, was specifically inactivated in the ureteric bud of the developing kidney. Mice lacking Mib1 in the renal collecting duct displayed increased urinary production, decreased urinary osmolality, progressive hydronephrosis, sodium wasting, and a severe urinary concentrating defect manifested as nephrogenic diabetes insipidus. Histological analysis revealed a diminished number of principal cells and corresponding increase in the number of intercalated cells. Transgenic overexpression of Notch intracellular domain reversed the altered cellular composition of mutant renal collecting duct, with principal cells occupying the entire region. Our data demonstrate that Notch signaling is required for the development of the mammalian renal collecting duct and principal cell differentiation and indicate that pathway dysregulation may contribute to distal renal tubular disorders.

Tonicity-responsive enhancer binding protein (TonEBP) plays a key role in protecting renal cells from hypertonic stress by stimulating transcription of specific genes. Under hypertonic conditions, TonEBP activity is enhanced via increased nuclear translocation, transactivation, and abundance. It was reported previously that hypertonicity exerted a dual, time-dependent effect on vasopressin-inducible aquaporin-2 (AQP2) expression in immortalized mouse collecting duct principal cells (mpkCCD cl4 ). Whereas AQP2 abundance decreased after 3 h of hyperosmotic challenge, it increased after 24 h of hypertonic challenge. This study investigated the role that TonEBP may play in these events by subjecting mpkCCD cl4 cells to 3 or 24 h of hypertonic challenge. Hypertonic challenge increased TonEBP mRNA and protein content and enhanced TonEBP activity as illustrated by both increased TonEBP-dependent luciferase activity and mRNA expression of several genes that are targeted by TonEBP. Irrespective of the absence or presence of vasopressin, decreased TonEBP activity in cells that were transfected with either TonEBP small interfering RNA or an inhibitory form of TonEBP strongly reduced AQP2 mRNA and protein content under iso-osmotic conditions and blunted the increase of AQP2 abundance that was induced after 24h of hypertonic challenge. Conversely, decreased TonEBP activity did not significantly alter reduced expression of AQP2 mRNA that was induced by 3 h of hypertonic challenge. Mutation of a TonE enhancer element located 489 bp upstream of the AQP2 transcriptional start site abolished the hypertonicity-induced increase of luciferase activity in cells that expressed AQP2 promoter-luciferase plasmid constructs, indicating that TonEBP influences AQP2 transcriptional activity at least partially by acting directly on the AQP2 promoter. These findings demonstrate that in collecting duct principal cells, TonEBP plays a central role in regulating AQP2 expression by enhancing AQP2 gene transcription.

Renal tubulo-interstitial inflammation is frequently associated with polyuria and urine concentration defects. This led us to investigate the effects of the major pro-inflammatory nuclear factor B (NF-B) pathway on aquaporin 2 (AQP2) expression by the collecting duct. Using immortalized collecting duct principal cells (mpkCCD cl4 ), we found that, acting independently of vasopressin, activation of NF-B by lipopolysaccharide (LPS) decreased AQP2 mRNA and protein levels in a time-and dose-dependent manner but did not decrease AQP2 mRNA stability. Consistently, constitutively active IB kinase ␤ decreased AQP2 expression. The LPSinduced decrease in AQP2 mRNA levels was confirmed in rat kidney slices and was reproduced both under conditions of elevated cAMP concentration and V 2 receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative B elements. Mutation of either B element abolished the LPS-induced decrease of luciferase activity in cells expressing AQP2 promoter-luciferase plasmid constructs. Chromatin immunoprecipitation revealed that LPS challenge decreased p65, increased p50 and p52, and had no effect on RelB and c-Rel binding to B elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-B subunits. We additionally found that hypertonicity activated NF-B in mpkCCD cl4 cells, an effect that may counteract the Tonicity-responsive enhancer binding protein (TonEBP)-dependent increase in AQP2 gene transcription. Taken together, these findings indicate that NF-B is an important physiological regulator of AQP2 transcription.Selective transcellular water permeability across the renal epithelium is facilitated by the aquaporin 1 (AQP1) 3 water channel, expressed in the proximal tubule and thin descending limb of Henle's loop (1, 2) and AQP2, -3, and -4, expressed in collecting duct (CD) principal cells (3). AQP2 inserted in the apical plasma membrane enhances CD apical water permeability. Water exits these cells via basolateral AQP3 and AQP4. By controlling both short term (plasma membrane insertion) and long term (transcriptional and post-transcriptional) AQP2 expression, the antidiuretic hormone [8-arginine]vasopressin (AVP) plays a major role in regulating water reabsorption (3-7). In addition to AVP, AQP2 expression is influenced by numerous factors that act independently of AVP including other hormones, i.e. aldosterone and insulin (8, 9), extracellular calcium (10), and environmental tonicity (11-13).The NF-B family of transcription factors consists of five members (p65, p50/p105, p52/p100, RelB, and c-Rel) that contain a Rel-homology domain required for the formation of various combinations of homo-and heterodimers and for DNA binding. Generally, dimers that contain p65 or c-Rel are transcriptional activators, whereas p50 and p52 homodimers act as repressors. The differential dimerization that occurs between NF-B family members allows for cellspecific transcriptional modulation of target genes in response t...

TonEBP stimulates genes whose products drive cellular accumulation of organic osmolytes and HSP70, which protect cells from the deleterious effects of hypertonicity and urea, respectively. Mice deficient in the TonEBP gene display severe atrophy of the renal medulla because cells failed to adapt to the hyperosmolality. Emerging data suggest that TonEBP plays a key role in the urinary concentrating mechanism by stimulating the UT-A urea transporters and possibly AQP2 water channel. Thus, TonEBP is an essential regulator in the urinary concentrating mechanism. Studies on structural basis of TonEBP function have revealed the structure of the DNA binding domain, and defined the transactivation domains. Molecular mechanisms underlying the nucleocytoplasmic trafficking, transactivation, and phosphorylation in response to changes in tonicity need to be understood in molecular detail. Such knowledge is needed for the identification of the sensor that detects changes in ambient tonicity and signals to TonEBP.

There is a significant immune response to ischemia-reperfusion injury (IRI), but the role of immunomodulatory natural killer T (NKT) cell subtypes is not well understood. Here, we compared the severity of IRI in mice deficient in type I/II NKT cells (CD1d

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