Altered expression of renal aquaporins and Na(+) transporters in rats treated with L-type calcium blocker

Nifedipine, a calcium antagonist, has diuretic and natriuretic properties. However, the molecular mechanisms by which these effects are produced are poorly understood. We examined kidney abundance of aquaporins (AQP1, AQP2, and AQP3) and major sodium transporters [type 3 Na/H exchanger (NHE-3); type 2 Na-Pi cotransporter (NaPi-2); Na-K-ATPase; type 1 bumetanide-sensitive cotransporter (BSC-1); and thiazide-sensitive Na-Cl cotransporter (TSC)] as well as inner medullary abundance of AQP2, phosphorylated-AQP2 (p-AQP2), AQP3, and calcium-sensing receptor (CaR). Rats treated with nifedipine orally (700 mg/kg) for 19 days had a significant increase in urine output, whereas urinary osmolality and solute-free water reabsorption were markedly reduced. Consistent with this, immunoblotting revealed a significant decrease in the abundance of whole kidney AQP2 (47 +/- 7% of control rats, P < 0.05) and in inner medullary AQP2 (60 +/- 7%) as well as in p-AQP2 abundance (17 +/- 6%) in nifedipine-treated rats. In contrast, whole kidney AQP3 abundance was significantly increased (219 +/- 28%). Of potential importance in modulating AQP2 levels, the abundance of CaR in the inner medulla was significantly increased (295 +/- 25%) in nifedipine-treated rats. Nifedipine treatment was also associated with increased urinary sodium excretion. Consistent with this, semiquantitative immunoblotting revealed significant reductions in the abundance of proximal tubule Na(+) transporters: NHE-3 (3 +/- 1%), NaPi-2 (53 +/- 12%), and Na-K-ATPase (74 +/- 5%). In contrast, the abundance of the distal tubule Na-Cl cotransporter (TSC) was markedly increased (240 +/- 29%), whereas BSC-1 in the thick ascending limb was not altered. In conclusion, 1) increased urine output and reduced urinary concentration in nifedipine-treated-rats may, in part, be due to downregulation of AQP2 and p-AQP2 levels; 2) CaR might be involved in the regulation of water reabsorption in the inner medulla collecting duct; 3) reduced expression of proximal tubule Na(+) transporters (NHE-3, NaPi-2, and Na, K-ATPase) may be involved in the increased urinary sodium excretion; and 4) increase in TSC expression may occur as a compensatory mechanism.     

4-1BB engagement costimulates NKT cell activation and exacerbates NKT cell ligand-induced airway hyperresponsiveness and inflammation

Multiple studies have demonstrated that 4-1BB (CD137), a member of the TNF receptor superfamily, is expressed on several immune cells including activated T cells. However, the expression and the role of 4-1BB on natural killer T (NKT) cells have not been fully characterized. In this study, it was shown that 4-1BB was not expressed on naive NKT cells but was rapidly induced on activated NKT cells by TCR engagement with alpha-galactosylceramide (alpha-GalCer). Also, 4-1BB signaling provided by 3H3, an agonistic anti-4-1BB mAb, promoted NKT cell activation resulting in enhanced cytokine production of NKT cells driven by alpha-GalCer. When NKT cell-driven airway immune responses were evaluated by intranasal administration of alpha-GalCer, airway hyperresponsiveness (AHR) and lung inflammation were significantly more aggravated in mice treated with 3H3 and alpha-GalCer than in mice treated with alpha-GalCer alone. These aggravations were accompanied by up-regulation of IL-4, IL-13, and IFN-gamma production. Interestingly, AHR was not developed in IL-4Ralpha-deficient mice treated with alpha-GalCer with or without 3H3 but was exacerbated in IFN-gamma-deficient mice. Our study suggests that 4-1BB on NKT cells functions as a costimulatory molecule and exacerbates the induction of NKT cell-mediated AHR, which is dependent on the IL-4Ralpha-mediated pathway.     

Increased expression of OsPT1, a high-affinity phosphate transporter, enhances phosphate acquisition in rice

Most high-affinity phosphate transporter genes (OsPTs) in rice were highly induced in roots when phosphate was depleted. OsPT1, however, was highly expressed in primary roots and leaves regardless of external phosphate concentrations. This finding was confirmed histochemically using transgenic rice plants that express the GUS reporter gene under the control of the OsPT1 promoter, which exhibited high GUS activity even in the phosphate sufficient condition. Furthermore, transgenic rice plants overexpressing the OsPT1 gene accumulated almost twice as much phosphate in the shoots as did wild-type plants. As a result, transgenic plants had more tillers than did wild-type plants, which is a typical physiological indicator for phosphate status in rice.     

LSGermOPA,a custom OPA of 384 EST-derived SNPs for high-throughput lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.) germplasm fingerprinting

To deploy a high-throughput genotyping platform in germplasm management, we designed and tested a custom OPA (Oligo Pool All), LSGermOPA, for assessing the genetic diversity and population structure of the USDA cultivated lettuce (Lactuca sativa L.) germplasm collection using Illumina’s GoldenGate assay. This OPA contains 384 EST (expressed sequence tag)-derived SNP (single nucleotide polymorphism) markers selected from a large set of SNP markers experimentally validated and mapped by the Compositae Genome Project. Used for genotyping were DNA samples prepared from bulked leaves of five randomly-selected seedlings from each of 380 lettuce accessions. High-quality genotype data were obtained from 354 of the 384 SNPs. The reproducibility of automatic genotype calls was 99.8% as calculated from the four pairs of duplicated DNA samples in the assay. An unexpectedly high percentage of heterozygous genotypes at the polymorphic loci for most accessions indicated a high level of heterogeneity within accessions. Only 148 homogenous accessions, collectively comprising all five horticultural types, were used in subsequent analyses to demonstrate the usefulness of LSGermOPA. The results of phylogenetic relationship, population structure and genetic differentiation analyses were consistent with previous reports using other marker systems. This suggests that LSGermOPA is capable of revealing sufficient levels of polymorphism among lettuce cultivars and is appropriate for rapid assessment of genetic diversity and population structure in the lettuce germplasm collection. Challenges and strategies for effective genotyping and managing lettuce germplasm are discussed.     

Functional interactions of meiotic recombination factors Rdh54 and Dmc1

Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54–Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces pombe proteins, we demonstrate complex formation between Rdh54 and Dmc1 and enhancement of the recombinase activity of Dmc1 by Rdh54. Consistent with published cytological and chromatin immunoprecipitation data that implicate Rdh54 in preventing the non-specific association of Dmc1 with chromatin, we show here that Rdh54 mediates the efficient removal of Dmc1 from dsDNA. These functional attributes of Rdh54 are reliant on its ATPase function. The results presented herein provide valuable information concerning the Rdh54–Dmc1 protein pair that is germane for understanding their role in meiotic recombination. The biochemical systems established in this study should be useful for the continuing dissection of the action mechanism of Rdh54 and Dmc1.