The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/ hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.
Proteolysis is an important part of protein identification in proteomics analysis. The conventional method of in-solution digestion of proteins is time-consuming and has limited sensitivity. In this study, trypsin- or alpha-chymotrypsin-immobilized microreactors prepared by a microfluidics-based enzyme-immobilization technique were studied for rapid sample preparation in proteomic analysis. The kinetic studies for hydrolysis of substrate by microreactors revealed that immobilized proteases had higher hydrolytic efficiency than those performed by in-solution digestion. The performance of the microreactors was evaluated by digesting cytochrome c and BSA. Protein digestion was achieved within a short period of time (approximately 5 min) at 30 degrees C without any complicated reduction and alkylation procedures. The efficiency of digestion by trypsin-immobilized reactor was evaluated by analyzing the sequence coverage, which was 47 and 12% for cytochrome c and BSA, respectively. These values were higher than those performed by in-solution digestion. Besides, because of higher stability against high concentration of denaturant, the microreactors can be useful for immediate digestion of the denaturated protein. In the present study, we propose a protease-immobilized microreactor digestion method, which can utilize as a proteome technique for biological and clinical research.
Advanced glycation end products (AGEs) and their receptor (RAGE) have a role in diabetic nephropathy. We have recently found that linagliptin, an inhibitor of dipeptidyl peptidase-4 (DPP-4), could inhibit renal damage in type 1 diabetic rats by suppressing the AGE-RAGE axis. However, it remains unclear whether DPP-4 deficiency could also have beneficial effects on experimental diabetic nephropathy. To address the issue, we rendered wild-type F344/NSlc and DPP-4-deficient F344/DuCrl/Crlj rats diabetic by injection of streptozotocin, and then investigated whether DPP-4 deficiency could block the activation of AGE-RAGE axis in the diabetic kidneys and resultantly ameliorate renal injury in streptozotocin-induced diabetic rats. Compared with control rats at 9 and 11 weeks old, body weight and heart rates were significantly lower, while fasting blood glucose was higher in wild-type and DPP-4-deficient diabetic rats at the same age. There was no significant difference of body weight, fasting blood glucose and lipid parameters between the two diabetic rat strains. AGEs, 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nitrotyrosine levels in the kidney, renal gene expression of RAGE and intercellular adhesion molecule-1, glomerular area, urinary excretion of 8-OHdG and albumin, and the ratio of renal to body weight were increased in wild-type diabetic rats at 9 and/or 11 weeks old compared with age-matched control rats, all of which except for urinary 8-OHdG levels at 11 weeks old were significantly suppressed in DPP-4-deficient diabetic rats. Our present study suggests that DPP-4 deficiency could exert beneficial actions on type 1 diabetic nephropathy partly by blocking the AGE-RAGE axis. DPP-4 might be a novel therapeutic target for preventing diabetic nephropathy. Sugars, including glucose and fructose, can react nonenzymatically with the amino groups of proteins, lipids and nucleic acids to form reversible Schiff bases, and then Amadori products. 1-3 These early glycation products undergo further complex reactions such as rearrangement, dehydration and condensation to become irreversibly cross-linked, heterogeneous fluorescent derivatives called 'advanced glycation end products (AGEs)'. 1-3 The process of non-enzymatic glycation is also known as Maillard reaction, and formation and accumulation of AGEs in various tissues have progressed at a physiological aging and at an extremely accelerated rate under diabetes. [1][2][3] There is a growing body of evidence that AGEs and their receptor (RAGE) interaction evoke oxidative stress generation and inflammatory and fibrotic reactions, thereby causing progressive alteration in renal architecture and loss of renal function in diabetes. [4][5][6][7][8] Furthermore, RAGEoverexpressing diabetic mice have been shown to exhibit progressive glomerulosclerosis with renal dysfunction, compared with diabetic littermates lacking the RAGE transgene. 9 Diabetic homozygous RAGE null mice displayed diminished albuminuria and glomerulosclerosis and failed to develop significantly increased mesan...
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