Gene Organization in Rice Revealed by Full-Length cDNA Mapping and Gene Expression Analysis through Microarray

Rice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE) genes, 33K annotated non-expressed (ANE) genes, and 5.5K non-annotated expressed (NAE) genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria

Urinary chemokine (C-C motif) ligand 2 (monocyte chemotactic protein-1) as a tubular injury marker for early detection of cisplatin-induced nephrotoxicity.

Because of the difficulty in detecting segment-specific response in the kidney, we investigated the molecular events underlying acute kidney injury in the proximal tubules of rats with cisplatin (cis-diamminedichloroplatinum II)-induced nephrotoxicity. Microarray analysis revealed that mRNA levels of several cytokines and chemokines, such as interleukin-1beta, chemokine (C-C motif) ligand (CCL) 2, CCL20, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL10 were significantly increased after cisplatin treatment in both isolated proximal tubules and whole kidney. Interestingly, tubular CCL2 mRNA levels increased soon after cisplatin administration, whereas CCL2 mRNA levels in whole kidney first decreased and then increased. Levels of both CCL2 and kidney injury molecule-1 (KIM-1) in the whole kidney increased after cisplatin administration. Immunofluorescence analysis revealed CCL2 changes in the proximal tubular cells initially and then in the medullary interstitium. Urinary CCL2 excretion significantly increased approximately 3-fold compared with controls the day after cisplatin administration (5mg/kg), when no changes were observed plasma creatinine and blood urea nitrogen levels. Urinary levels of KIM-1 also increased 3-fold after cisplatin administration. In addition, urinary CCL2 rather than KIM-1 increased in chronic renal failure rats after administration of low-dose cisplatin (2mg/kg), suggesting that urinary CCL2 was selective for cisplatin-induced nephrotoxicity in renal impairment. These results indicated that the increase in cytokine and chemokine expression in renal epithelial cells might be responsible for kidney deterioration in cisplatin-induced nephrotoxicity, and that urinary CCL2 is associated with tubular injury and serves as a sensitive and noninvasive marker for the early detection of cisplatin-induced tubular injury

Ezrin, a membrane cytoskeletal cross-linker, is essential for the regulation of phosphate and calcium homeostasis

Ezrin cross-links plasma membrane proteins with the actin cytoskeleton. In the kidney, ezrin mainly localizes at the brush border membrane of proximal tubules with the scaffolding protein, Na+/H+ exchanger regulatory factor (NHERF) 1. NHERF1 interacts with the sodium/phosphate cotransporter, Npt2a. Defects in NHERF1 or Npt2a in mice cause hypophosphatemia. Here we studied the physiological role of ezrin in renal phosphate reabsorption using ezrin knockdown mice (Vil2). These mice exhibit hypophosphatemia, hypocalcemia, and osteomalacia. The reduced plasma phosphate concentrations were ascribed to defects in urinary phosphate reabsorption. Immunofluorescence and immunoblotting indicated a marked reduction in renal Npt2a and NHERF1 expression at the apical membrane of proximal tubules in the knockdown mice. On the other hand, urinary loss of calcium was not found in Vil2 mice. Plasma concentrations of 1,25-dihydroxyvitamin D were elevated following reduced plasma phosphate levels, and mRNA of the vitamin D–dependent TRPV6 calcium channel were significantly increased in the duodenum of knockdown mice. Expression of TRPV6 at the apical membrane, however, was significantly decreased. Furthermore, tibial bone mineral density was significantly lower in both the adult and young Vil2 mice. These results suggest that ezrin is required for the regulation of systemic phosphate and calcium homeostasis in vivo