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.

SummaryA glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs : orf1 , orf2 and orf3 . Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, D orf1 and D orf2 , exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from D orf1 mutant using aspartic N -peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the D orf1 , D orf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22 phox and gp91 phox , membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22 PHOX and gp91 PHOX. This led to blockade of the AGE-elicited Ras activation and NF-B-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidasemediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy.

The present study suggests that AGE-RAGE-mediated ROS generation activates TGF-beta-Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.

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