Transcription Factors in Alkaloid Engineering

Plants produce a large variety of low-molecular-weight and specialized secondary compounds. Among them, nitrogen-containing alkaloids are the most biologically active and are often used in the pharmaceutical industry. Although alkaloid chemistry has been intensively investigated, characterization of alkaloid biosynthesis, including biosynthetic enzyme genes and their regulation, especially the transcription factors involved, has been relatively delayed, since only a limited number of plant species produce these specific types of alkaloids in a tissue/cell-specific or developmental-specific manner. Recent advances in molecular biology technologies, such as RNA sequencing, co-expression analysis of transcripts and metabolites, and functional characterization of genes using recombinant technology and cutting-edge technology for metabolite identification, have enabled a more detailed characterization of alkaloid pathways. Thus, transcriptional regulation of alkaloid biosynthesis by transcription factors, such as basic helix–loop–helix (bHLH), APETALA2/ethylene-responsive factor (AP2/ERF), and WRKY, is well elucidated. In addition, jasmonate signaling, an important cue in alkaloid biosynthesis, and its cascade, interaction of transcription factors, and post-transcriptional regulation are also characterized and show cell/tissue-specific or developmental regulation. Furthermore, current sequencing technology provides more information on the genome structure of alkaloid-producing plants with large and complex genomes, for genome-wide characterization. Based on the latest information, we discuss the application of transcription factors in alkaloid engineering

Effects of CEOs' Characteristics on Internationalization of Small and Medium Enterprises in Japan

Recent heterogeneous-firm models of international trade suggest that productivity determines whether firms engage in export and foreign direct investment. However in practice, m Abstract any productive firms are not internationalized, whereas many unproductive firms are. This situation suggests that factors other than productivity influence internationalization. This study examines a set of potential factors -personal characteristics of the chief executive officer (CEO)- using a unique panel dataset for Japanese small and medium enterprises (SMEs). We find that SMEs are more likely to be internationalized when the CEO is more risk-tolerant, forward-looking, and internationally experienced. These factors show significant statistical relationships with SMEs' decisions to internationalize, perhaps suggesting why productive firms might not internationalize. In addition, we find that productivity has no significant relationship with the decision of exiting international markets probably because initial costs of internationalization become sunk, whereas SMEs with internationally experienced CEOs show strongly less likelihood of exit. These empirical results are consistent with theoretical predictions of our model that incorporates the uncertainty of foreign markets into the trade theory with firm heterogeneity.

Temporal pattern of loss/persistence of duplicate genes involved in signal transduction and metabolic pathways after teleost-specific genome duplication

<p>Abstract</p> <p>Background</p> <p>Recent genomic studies have revealed a teleost-specific third-round whole genome duplication (3R-WGD) event occurred in a common ancestor of teleost fishes. However, it is unclear how the genes duplicated in this event were lost or persisted during the diversification of teleosts, and therefore, how many of the duplicated genes contribute to the genetic differences among teleosts. This subject is also important for understanding the process of vertebrate evolution through WGD events. We applied a comparative evolutionary approach to this question by focusing on the genes involved in long-term potentiation, taste and olfactory transduction, and the tricarboxylic acid cycle, based on the whole genome sequences of four teleosts; zebrafish, medaka, stickleback, and green spotted puffer fish.</p> <p>Results</p> <p>We applied a state-of-the-art method of maximum-likelihood phylogenetic inference and conserved synteny analyses to each of 130 genes involved in the above biological systems of human. These analyses identified 116 orthologous gene groups between teleosts and tetrapods, and 45 pairs of 3R-WGD-derived duplicate genes among them. This suggests that more than half [(45×2)/(116+45)] = 56.5%) of the loci, probably more than ten thousand genes, present in a common ancestor of the four teleosts were still duplicated after the 3R-WGD. The estimated temporal pattern of gene loss suggested that, after the 3R-WGD, many (71/116) of the duplicated genes were rapidly lost during the initial 75 million years (MY), whereas on average more than half (27.3/45) of the duplicated genes remaining in the ancestor of the four teleosts (45/116) have persisted for about 275 MY. The 3R-WGD-derived duplicates that have persisted for a long evolutionary periods of time had significantly larger number of interacting partners and longer length of protein coding sequence, implying that they tend to be more multifunctional than the singletons after the 3R-WGD.</p> <p>Conclusion</p> <p>We have shown firstly the temporal pattern of gene loss process after 3R-WGD on the basis of teleost phylogeny and divergence time frameworks. The 3R-WGD-derived duplicates have not undergone constant exponential decay, suggesting that selection favoured the long-term persistence of a subset of duplicates that tend to be multi-functional. On the basis of these results obtained from the analysis of 116 orthologous gene groups, we propose that more than ten thousand of 3R-WGD-derived duplicates have experienced lineage-specific evolution, that is, the differential sub-/neo-functionalization or secondary loss between lineages, and contributed to teleost diversity.</p

Genome-wide identification of AP2/ERF transcription factor-encoding genes in California poppy (Eschscholzia californica) and their expression profiles in response to methyl jasmonate

With respect to the biosynthesis of plant alkaloids, that of benzylisoquinoline alkaloids (BIAs) has been the most investigated at the molecular level. Previous investigations have shown that the biosynthesis of BIAs is comprehensively regulated by WRKY and bHLH transcription factors, while promoter analyses of biosynthesis enzyme-encoding genes have also implicated the involvement of members of the APETALA2/ethylene responsive factor (AP2/ERF) superfamily. To investigate the physiological roles of AP2/ERF transcription factors in BIA biosynthesis, 134 AP2/ERF genes were annotated using the draft genome sequence data of Eschscholzia californica (California poppy) together with transcriptomic data. Phylogenetic analysis revealed that these genes could be classified into 20 AP2, 5 RAV, 47 DREB, 60 ERF and 2 Soloist family members. Gene structure, conserved motif and orthologous analyses were also carried out. Gene expression profiling via RNA sequencing in response to methyl jasmonate (MeJA) indicated that approximately 20 EcAP2/ERF genes, including 10 group IX genes, were upregulated by MeJA, with an increase in the expression of the transcription factor-encoding gene EcbHLH1 and the biosynthesis enzyme-encoding genes Ec6OMT and EcCYP719A5. Further quantitative RT-PCR confirmed the MeJA responsiveness of the EcAP2/ERF genes, i.e., the increased expression of 9 group IX, 2 group X and 2 group III ERF subfamily genes. Transactivation activity of group IX EcAP2/ERFs was also confirmed by a luciferase reporter assay in conjunction with the promoters of the Ec6OMT and EcCYP719A5 genes. The physiological roles of AP2/ERF genes in BIA biosynthesis and their evolution in the regulation of alkaloid biosynthesis are discussed

Genome-Wide Profiling of WRKY Genes Involved in Benzylisoquinoline Alkaloid Biosynthesis in California Poppy (Eschscholzia californica)

Transcription factors of the WRKY family play pivotal roles in plant defense responses, including the biosynthesis of specialized metabolites. Based on the previous findings of WRKY proteins regulating benzylisoquinoline alkaloid (BIA) biosynthesis, such as CjWRKY1—a regulator of berberine biosynthesis in Coptis japonica—and PsWRKY1—a regulator of morphine biosynthesis in Papaver somniferum—we performed genome-wide characterization of the WRKY gene family in Eschscholzia californica (California poppy), which produces various BIAs. Fifty WRKY genes were identified by homology search and classified into three groups based on phylogenetic, gene structure, and conserved motif analyses. RNA sequencing showed that several EcWRKY genes transiently responded to methyl jasmonate, a known alkaloid inducer, and the expression patterns of these EcWRKY genes were rather similar to those of BIA biosynthetic enzyme genes. Furthermore, tissue expression profiling suggested the involvement of a few subgroup IIc EcWRKYs in the regulation of BIA biosynthesis. Transactivation analysis using luciferase reporter genes harboring the promoters of biosynthetic enzyme genes indicated little activity of subgroup IIc EcWRKYs, suggesting that the transcriptional network of BIA biosynthesis constitutes multiple members. Finally, we investigated the coexpression patterns of EcWRKYs with some transporter genes and discussed the diversified functions of WRKY genes based on a previous finding that CjWRKY1 overexpression in California poppy cells enhanced BIA secretion into the medium

Place of pitavastatin in the statin armamentarium: promising evidence for a role in diabetes mellitus

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, known as statins, have revolutionized the treatment of hypercholesterolemia and coronary artery disease prevention. However, there are considerable issues regarding statin safety and further development of residual risk control, particularly for diabetic and metabolic syndrome patients. Pitavastatin is a potent statin with low-density lipoprotein (LDL) cholesterol-lowering effects comparable to those of atorvastatin or rosuvastatin. Pitavastatin has a high-density lipoprotein (HDL) cholesterol raising effect, may improve insulin resistance, and has little influence on glucose metabolism. Considering these factors along with its unique pharmacokinetic properties, which suggest minimal drug–drug interaction, pitavastatin could provide an alternative treatment choice, especially in patients with glucose intolerance or diabetes mellitus. Many clinical trials are now underway to test the clinical efficacy of pitavastatin in various settings and are expected to provide further information