Modification of genes through homologous recombination, termed gene targeting, is the most direct method to characterize gene function. In higher plants, however, the method is far from a common practice. Here we describe an efficient and reproducible procedure with a strong positive/negative selection for gene targeting in rice, which feeds more than half of the world's population and is an important model plant. About 1% of selected calli and their regenerated fertile plants were heterozygous at the targeted locus, and only one copy of the selective marker used was found at the targeted site in their genomes. The procedure's applicability to other genes will make it feasible to obtain various gene-targeted lines of rice.

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The impact of AGEs on human MSCs was studied. AGEs inhibited the proliferation of MSCs, induced apoptosis, and prevented cognate differentiation into adipose tissue, cartilage, and bone, suggesting a deleterious effect of AGEs in the pathogenesis of musculoskeletal disorders in aged and diabetic patients.Introduction: Advanced glycation end-products (AGEs) are accumulated on long-lived proteins of various tissues in advanced age and diabetes mellitus and have been implicated in chronic complication, including musculoskeletal disorders. Human mesenchymal stem cells (MSCs) potentially differentiate into mature musculoskeletal tissues during tissue repair, but the pathogenetic role of AGEs on MSCs is unclear. Materials and Methods: AGEs were prepared by incubating BSA with glucose, glyceraldehydes, or glycolaldehyde (designated as AGE-1, AGE-2, or AGE-3, respectively). Proliferation, apoptosis, and reactive oxygen species (ROS) generation were assayed in AGE-treated cells. The expression of the receptor for AGE (RAGE) was examined by immunohistochemistry and Western blotting. Involvement of RAGE-mediated signaling was examined using a neutralizing antiserum against RAGE. Differentiation into adipose tissue, cartilage, and bone were morphologically and biochemically monitored with specific markers for each. Results: AGE-2 and AGE-3, but not control nonglycated BSA and AGE-1, reduced the viable cell number and 5-bromo-2'deoxyuridine (BrdU) incorporation with increased intracellular ROS generation and the percentage of apoptotic cells. MSCs expressed RAGE and its induction was stimulated by AGE-2 and AGE-3. These AGEs inhibited adipogenic differentiation (assayed by oil red O staining, lipoprotein lipase production, and intracellular triglyceride content) and chondrogenic differentiation (assayed by safranin O staining and type II collagen production). On osteogenic differentiation, AGE-2 and AGE-3 increased alkaline phosphatase activity and intracellular calcium content; however, von Kossa staining revealed the loss of mineralization and mature bone nodule formation. The antiserum against RAGE partially prevented AGE-induced cellular events. Conclusion: AGE-2 and AGE-3 may lead to the in vivo loss of MSC mass and the delay of tissue repair by inhibiting the maturation of MSC-derived cells. The AGE-RAGE interaction may be involved in the deleterious effect of AGEs on MSCs.

We previously have found that advanced glycation end products (AGE), senescent macroproteins formed at an accelerated rate in diabetes, arise in vivo not only from glucose but also from reducing sugars. Furthermore, we recently have shown that glyceraldehyde- and glycolaldehyde-derived AGE (glycer- and glycol-AGE) are mainly involved in loss of pericytes, the earliest histopathological hallmark of diabetic retinopathy. However, the effects of these AGE proteins on angiogenesis, another vascular derangement in diabetic retinopathy, remain to be elucidated. In this study, we investigated whether these AGE proteins elicit changes in cultured endothelial cells that are associated with angiogenesis. When human skin microvascular endothelial cells (EC) were cultured with glycer-AGE or glycol-AGE, growth and tube formation of EC, the key steps of angiogenesis, were significantly stimulated. The AGE-induced growth stimulation was significantly enhanced in AGE receptor (RAGE)-overexpressed EC. Furthermore, AGE increased transcriptional activity of nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) and then up-regulated mRNA levels of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in EC. Cerivastatin, a hydroxymethylglutaryl CoA reductase inhibitor; pyrrolidinedithiocarbamate; or curcumin was found to completely prevent the AGE-induced increase in NF-kB and AP-1 activity, VEGF mRNA up-regulation, and the resultant increase in DNA synthesis in microvascular EC. These results suggest that the AGE-RAGE interaction elicited angiogenesis through the transcriptional activation of the VEGF gene via NF-kB and AP-1 factors. By blocking AGE-RAGE signaling pathways, cerivastatin might be a promising remedy for treating patients with proliferative diabetic retinopathy.