Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.     

Crystal structure of human cytosolic aspartyl‐tRNA synthetase,a component of multi‐tRNA synthetase complex

Human cytosolic aspartyl‐tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi‐tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Ų which comprises 16.6% of the monomeric surface area. Our structure reveals the C‐terminal end of the N‐helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N‐helix for the transfer of tRNA^Asp to elongation factor 1α. From our analyses of the crystal structure and post‐translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.Proteins 2013; 81:1840–1846. © 2013 Wiley Periodicals, Inc.     

Gastrokine 1 regulates NF‐κB signaling pathway and cytokine expression in gastric cancers

Gastrokine 1 (GKN1) plays an important role in the gastric mucosal defense mechanism and also acts as a functional gastric tumor suppressor. In this study, we examined the effect of GKN1 on the expression of inflammatory mediators, including NF‐κB, COX‐2, and cytokines in GKN1‐transfected AGS cells and shGKN1‐transfected HFE‐145 cells. Lymphocyte migration and cell viability were also analyzed after treatment with GKN1 and inflammatory cytokines in AGS cells by transwell chemotaxis and an MTT assay, respectively. In GKN1‐transfected AGS cells, we observed inactivation and reduced expression of NF‐κB and COX‐2, whereas shGKN1‐transfected HFE‐145 cells showed activation and increased expression of NF‐κB and COX‐2. GKN1 expression induced production of inflammatory cytokines including IL‐8 and ‐17A, but decreased expression of IL‐6 and ‐10. We also found IL‐17A expression in 9 (13.6%) out of 166 gastric cancer tissues and its expression was closely associated with GKN1 expression. GKN1 also acted as a chemoattractant for the migration of Jurkat T cells and peripheral B lymphocytes in the transwell assay. In addition, GKN1 significantly reduced cell viability in both AGS and HFE‐145 cells. These data suggest that the GKN1 gene may inhibit progression of gastric epithelial cells to cancer cells by regulating NF‐κB signaling pathway and cytokine expression. J. Cell. Biochem. 114: 1800–1809, 2013. © 2013 Wiley Periodicals, Inc.     

Arabidopsis Brassinosteroid-overproducing gulliver3-D/dwarf4-D mutants exhibit altered responses to Jasmonic acid and pathogen

Key message

Arabidopsis gulliver3 - D/dwarf4 - D displays growth-promoting phenotypes due to activation tagging of a key brassinosteroid biosynthetic gene DWARF4. In gul3-D/dwf4-D , the Jasmonate and Salicylate signaling pathways were relatively activated and suppressed, respectively.

Abstract

Energy allocation between growth and defense is elegantly balanced to achieve optimal development in plants. Brassinosteroids (BRs), steroidal hormones essential for plant growth, are regulated by other plant hormones, including auxin and jasmonates (JA); auxin stimulates the expression of a key brassinosteroid (BR) biosynthetic gene, DWARF4 (DWF4), whereas JA represses it. To better understand the interaction mechanisms between growth and defense, we isolated a fast-growing mutant, gulliver3-D (gul3-D), that resulted from the activation tagging of DWF4, and examined the response of this mutant to defense signals, including JA, Pseudomonas syringae pv. tomato (Pst DC3000) infection, and wounding. The degree of root growth inhibition following MeJA treatment was significantly decreased in gul3-1D/dwf4-5D relative to the wild type, suggesting that JA signaling is partially desensitized in gul3-1D. Quantitative RT-PCR analysis of the genes involved in JA and salicylic acid (SA) responses, including MYC2, PDF1.2, CORI3, PR1, and PR2, revealed that JA signaling was preferentially activated in gul3-1D, whereas SA signaling was suppressed. As a result, gul3-1D was more susceptible to a biotrophic pathogen, Pst DC3000. Based on our results, we propose a model in which BR and JA cooperate to balance energy allocation between growth and defense responses. In ambient conditions, BRs promote plant growth; however, when stresses trigger JA signaling, JA compromises BR signaling by downregulating DWF4 expression.     

Effect of Zinc Oxide Nanoparticles on the Function of MC3T3-E1 Osteoblastic Cells

Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 μg/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01～1 μg/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 μg/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells (P?<?0.05). Moreover, pretreatment with ZnO NPs (0.01～1 μg/ml) significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Measurement of reactive oxygen species (ROS) indicated decrease in ROS level upon exposure to ZnO nanoparticles (0.01 μg/ml). Hence, our study indicated that ZnO nanoparticles can have protective effects on osteoblasts at low concentrations where there are little or no observable cytotoxic effects.     

A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater.