Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene

Background: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds. Results: Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds. Conclusion: We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries

A Case of Metastatic Extramammary Paget's Disease Responding to Trastuzumab plus Paclitaxel Combination Therapy

There is no effective treatment for advanced extramammary Paget's disease (EMPD). The human epidermal growth factor receptor 2 (HER2) protein is often overexpressed in EMPD. Trastuzumab is a humanized monoclonal antibody against HER2 used in the treatment of breast cancers in which HER2 is overexpressed. We report a case of advanced EMPD in which trastuzumab and paclitaxel combination therapy was effective. The patient was a 70-year-old Japanese woman who presented with EMPD on the vulva and multiple metastatic lymph nodes. Immunohistochemical staining revealed strong HER2 protein expression in the primary tumor and metastatic lymph nodes. The patient received trastuzumab and paclitaxel. After 4 courses of this regimen, the mass on the vulva and the metastatic lymph nodes regressed. Our findings may imply that trastuzumab plus paclitaxel combination therapy is useful for the treatment of advanced EMPD overexpressing HER2

Loss of ATF6α in a Human Carcinoma Cell Line Is Compensated not by Its Paralogue ATF6β but by Sustained Activation of the IRE1 and PERK Arms for Tumor Growth in Nude Mice

To survive poor nutritional conditions, tumor cells activate the unfolded protein response, which is composed of the IRE1, PERK, and ATF6 arms, to maintain the homeostasis of the endoplasmic reticulum, where secretory and transmembrane proteins destined for the secretory pathway gain their correct three-dimensional structure. The requirement of the IRE1 and PERK arms for tumor growth in nude mice is established. Here we investigated the requirement for the ATF6 arm, which consists of ubiquitously expressed ATF6α and ATF6β, by constructing ATF6α-knockout (KO), ATF6β-KO, and ATF6α/β-double KO (DKO) in HCT116 cells derived from human colorectal carcinoma. Results showed that these KO cells grew similarly to wild-type (WT) cells in nude mice, contrary to expectations from our analysis of ATF6α-KO, ATF6β-KO, and ATF6α/β-DKO mice. We then found that the loss of ATF6α in HCT116 cells resulted in sustained activation of the IRE1 and PERK arms in marked contrast to mouse embryonic fibroblasts, in which the loss of ATF6α is compensated for by ATF6β. Although IRE1-KO in HCT116 cells unexpectedly did not affect tumor growth in nude mice, IRE1-KO HCT116 cells with ATF6α knockdown grew significantly more slowly than WT or IRE1-KO HCT116 cells. These results have unraveled the situation-dependent differential compensation strategies of ATF6α

Global Structure of Moduli Space for BPS Walls

We study the global structure of the moduli space of BPS walls in the Higgs branch of supersymmetric theories with eight supercharges. We examine the structure in the neighborhood of a special Lagrangian submanifold M, and find that the dimension of the moduli space can be larger than that naively suggested by the index theorem, contrary to previous examples of BPS solitons. We investigate BPS wall solutions in an explicit example of M using Abelian gauge theory. Its Higgs branch turns out to contain several special Lagrangian submanifolds including M. We show that the total moduli space of BPS walls is the union of these submanifolds. We also find interesting dynamics between BPS walls as a byproduct of the analysis. Namely, mutual repulsion and attraction between BPS walls sometimes forbid a movement of a wall and lock it in a certain position; we also find that a pair of walls can transmute to another pair of walls with different tension after they pass through.Comment: 42 pages, 11 figures; a few comments adde

Induction and Enhancement of Cardiac Cell Differentiation from Mouse and Human Induced Pluripotent Stem Cells with Cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1+ common progenitor cells, and identified highly cardiogenic progenitors as Flk1+/CXCR4+/VE-cadherin− (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1+ cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1+ cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs

Induction of Liver Steatosis and Lipid Droplet Formation in ATF6α-Knockout Mice Burdened with Pharmacological Endoplasmic Reticulum Stress

We burdened mice with intraperitoneal injection of the endoplasmic reticulum stress-inducing reagent tunicamycin, and found that wild-type mice were able to recover from the insult, whereas ATF6α-knockout mice exhibited liver dysfunction and steatosis. Our results establish links between endoplasmic reticulum stress, lipid metabolism and steatosi

Introduction to the SPARC Reanalysis Intercomparison Project (S-RIP) and overview of the reanalysis systems

The climate research community uses atmospheric reanalysis data sets to understand a wide range of processes and variability in the atmosphere, yet different reanalyses may give very different results for the same diagnostics. The Stratosphere–troposphere Processes And their Role in Climate (SPARC) Reanalysis Intercomparison Project (S-RIP) is a coordinated activity to compare reanalysis data sets using a variety of key diagnostics. The objectives of this project are to identify differences among reanalyses and understand their underlying causes, to provide guidance on appropriate usage of various reanalysis products in scientific studies, particularly those of relevance to SPARC, and to contribute to future improvements in the reanalysis products by establishing collaborative links between reanalysis centres and data users. The project focuses predominantly on differences among reanalyses, although studies that include operational analyses and studies comparing reanalyses with observations are also included when appropriate. The emphasis is on diagnostics of the upper troposphere, stratosphere, and lower mesosphere. This paper summarizes the motivation and goals of the S-RIP activity and extensively reviews key technical aspects of the reanalysis data sets that are the focus of this activity. The special issue The SPARC Reanalysis Intercomparison Project (S-RIP) in this journal serves to collect research with relevance to the S-RIP in preparation for the publication of the planned two (interim and full) S-RIP reports