Characterization of three Arabidopsis homologs of human RING membrane anchor E3 ubiquitin ligase

Ubiquitination affects diverse physiological processes in eukaryotic cells. AtRMA1 was previously identified as an Arabidopsis homolog of human RING membrane-anchor E3 ubiquitin (Ub) ligase. Here, we identified two additional AtRMA homologs, AtRMA2 and AtRMA3. The predicted AtRMA proteins contain a RING motif and a trans-membrane domain in their N-terminal and extreme C-terminal regions, respectively. Bacterially expressed AtRMAs exhibited E3 ligase activity in vitro, which was abrogated by mutation of the conserved cysteine residue in their RING domains. In vivo targeting experiments using an Arabidopsis protoplast-transfection system showed that all three AtRMAs are localized to the ER. Although RT-PCR analysis indicated that AtRMA mRNAs were expressed constitutively in all tissues examined, their promoter activities were differentially detected in a tissue-specific fashion in AtRMA-promoter::GUS transgenic Arabidopsis plants. The AtRMA1 and AtRMA3 genes are predominantly expressed in major tissues, such as cotyledons, leaves, shoot–root junction, roots, and anthers, while AtRMA2 expression is restricted to the root tips and leaf hydathodes. We suggest that a ubiquitnation pathway involving these AtRMA E3 Ub ligases may play a role in the growth and development of Arabidopsis.     

Pinoresinol-4,4′-di-O-β-d-glucoside from Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse embryo fibroblasts

Lignans are major constituents of plant extracts and have important pharmacological effects on mammalian cells. Here we showed that pinoresinol-4,4′-di-O-β-d-glucoside (PDG) from Valeriana officinalis induced calcium mobilization and cell migration through the activation of lysophosphatidic acid (LPA) receptor subtypes. Stimulation of mouse embryo fibroblast (MEF) cells with 10 μM PDG resulted in strong stimulation of MEF cell migration and the EC₅₀ was about 2 μM. Pretreatment with pertussis toxin (PTX), an inhibitor of G_i protein, completely blocked PDG-induced cell migration demonstrating that PDG evokes MEF cell migration through the activation of the G_i-coupled receptor. Furthermore, pretreatment of MEF cells with Ki16425 (10 μM), which is a selective antagonist for LPA₁ and LPA₃ receptors, completely blocked PDG-induced cell migration. Likewise, PDG strongly induced calcium mobilization, which was also blocked by Ki16425 in a dose-dependent manner. Prior occupation of the LPA receptor with LPA itself completely blocked PDG-induced calcium mobilization. Finally, PDG-induced MEF cell migration was attenuated by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor such as LY294002. Cells lacking downstream mediator of PI3K such as Akt1 and Akt2 (DKO cells) showed loss of PDG-induced migration. Re-expression of Akt1 (but not Akt2) completely restored PDG-induced DKO cell migration. Given these results, we conclude that PDG is a strong inducer of cell migration. We suggest that the pharmacological action of PDG may occur through the activation of an LPA receptor whereby activation of PI3K/Akt signaling pathway mediates PDG-induced MEF cell migration.     

Sequence diversity of a domesticated transposase gene, <Emphasis Type="Italic">MUG1</Emphasis>, in <Emphasis Type="Italic">Oryza</Emphasis> species

MUG1 is a MULE transposon-related domesticated gene in plants. We assessed the sequence diversity, neutrality, expression, and phylogenetics of the MUG1 gene among Oryza ssp. We found MUG1 expression in all tissues analyzed, with different levels in O. sativa. There were 408 variation sites in the 3886 bp of MUG1 locus. The nucleotide diversity of the MUG1 was higher than functionally known genes in rice. The nucleotide diversity (π) in the domains was lower than the average nucleotide diversity in whole coding region. The π values in nonsynonymous sites were lower than those of synonymous sites. Tajima D and Fu and Li D* values were mostly negative values, suggesting purifying selection in MUG1 sequences of Oryza ssp. Genome-specific variation and phylogenetic analyses show a general grouping of MUG1 sequences congruent with Oryza ssp. biogeography; however, our MUG1 phylogenetic results, in combination with separate B and D genome studies, might suggest an early divergence of the Oryza ssp. by continental drift of Gondwanaland. O. longistaminata MUG1 divergence from other AA diploids suggests that it might not be a direct ancestor of the African rice species. These authors contributed equally to this work.     

Molecular evidence for the hybridity ofIlex × wandoensis and the phylogenetic study of KoreanIlex based on ITS sequence data

In this study, internal transcribed spacer (ITS) regions within the nuclear ribosomal DNA of KoreanIlex were analyzed in order to investigate any molecular evidence thatI. × wandoensis could serve as a putative hybrid betweenI. cornuta andI. integra. We also sought to clearly identify taxonomic relationships and problems caused by consecutive external morphological characters among taxa in the genus. We sequenced 20 clones fromI. × wandoensis and found these individuals displayed intra-genomic polymorphisms within ITS regions. The analysis of the clones clearly demonstrated the presence of discrete sequences from bothI. cornuta andI. integra, thereby confirmingI. × wandoensis is a species that was formed by crossingI. cornuta andI. integra. Lastly, the subgenusIlex, which contains the evergreen species, failed to form a monophyletic group in a strict consensus tree that was prepared based upon ITS regions.I. crenata var.microphylla in the subgenus KoreaIlex, which has presented taxonomic problems previously, formed an independent clade within the consensus tree, thereby showing distinction fromI. crenata.) Genetic discontinuity ofI. macropoda andI. Macropoda for.pseudo-macropoda individuals couldn't be confirmed.     

Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B

Insulin is a polypeptide hormone which is produced by the β‐cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des‐threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des‐threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2–3 at 25 °C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des‐threonine insulin and hence increased the production yield of active insulin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Molecular determinants of the interactions between LXR/RXR heterodimers and TRAP220

Dimerization-induced activation of LXR is a unique allosteric mechanism described only for LXR/RXR heterodimers. Previously, we demonstrated that RXR functions as an allosteric activator of LXR binding to ASC-2 coactivator rather than as a direct interaction partner. Here, we investigated the molecular basis of the interaction between LXR/RXR and TRAP220 fragment (TN1/2) harboring two NR boxes. We found that either LXR binding to NR box-2 or RXR binding to NR box-1 was sufficient for optimal LXR/RXR binding to TN1/2, indicating that both receptors contribute equally in this interaction. Notably, the AF2 deletion of either receptor completely abolished LXR/RXR-TN1/2 interaction, suggesting dual roles for both AF2 domains in direct interaction with target NR boxes as well as in allosteric activation of partner receptors. We also found specific residues within NR box-2 required for LXR binding using one- plus two-hybrid system and identified Pro⁶⁴³ residue as a major determinant for NR specificity.     

Random amplified polymorphic DNA analysis of genetically modified organisms

Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.