Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches

Correlations Between Serum Cytokine Levels and the Use of a Moisturizer in Elderly Women in Accordance with the Improvement of Objective and Subjective Skin Condition

Miki Iwai,1 Koichi Nakaoji,1 Kazuhiko Hamada,1 Yutaka Inaba,2 Kyoko Muraoka,2 Emi Tohsuji,2 Masatoshi Jinnin2 1Central R&D Laboratory, Pias Corporation, Kobe, Japan; 2Department of Dermatology, Wakayama Medical University, Wakayama, JapanCorrespondence: Miki Iwai, Central R&D Laboratory, Pias Corporation, 1-3-1, Murotani, Nishi-ku, Kobe, 651-2241, Japan, Tel +81-78-992-6591, Fax +81-78-992-6556, Email [email protected]: In the skin of elderly people with dryness, the production of inflammatory cytokines tends to be induced under the influence of external stimuli. Therefore, there has been a hypothesis that the deterioration of skin conditions due to aging is linked to systemic inflammation. This study aimed to verify the possibility that the use of moisturizer improves skin condition and suppresses systemic inflammation.Methods: As an open study, the participants (n=75) were randomly assigned to either control group or moisturizer group. Participants in the moisturizer group used a moisturizer called Grafa Moisture Keep Milk MC at least twice a day for four weeks on the entire body below the neck. Objective skin conditions (overall dry skin score, water content of the stratum corneum, and transepidermal water loss) and serum cytokine levels (IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8, and TNF-α) were evaluated before and after the study in both groups. Subjective skin condition (questionnaire evaluation) was also assessed in the moisturizer group after the study.Results: Serum IL-6 level was significantly reduced in the moisturizer group (n=16) compared with the control group (n=36). In addition, there was an inverse correlation between serum IL-5 and the subjective moisturizing effect in the questionnaire evaluation, suggesting that the moisturizer improved subjective symptoms of dryness by reducing IL-5 levels. Furthermore, there was a positive correlation between IL-5 and IL-6, indicating that they are regulated by common upstream factors. A significant positive correlation of transepidermal water loss with serum IL-4 levels was also detected.Conclusion: The application of the moisturizer to the entire body not only improved subjective and objective skin condition, it may also reduce the levels of circulating inflammatory cytokines.Umin Clinical Trials Registry: Registration number: UMIN 000052024.Keywords: aging, cytokine, dryness, inflammation, skin car

Cinnamtannin B-1 Promotes Migration of Mesenchymal Stem Cells and Accelerates Wound Healing in Mice

<div><p>Substances that enhance the migration of mesenchymal stem cells to damaged sites have the potential to improve the effectiveness of tissue repair. We previously found that ethanol extracts of <i>Mallotus philippinensis</i> bark promoted migration of mesenchymal stem cells and improved wound healing in a mouse model. We also demonstrated that bark extracts contain cinnamtannin B-1, a flavonoid with <i>in vitro</i> migratory activity against mesenchymal stem cells. However, the <i>in vivo</i> effects of cinnamtannin B-1 on the migration of mesenchymal stem cells and underlying mechanism of this action remain unknown. Therefore, we examined the effects of cinnamtannin B-1 on <i>in vivo</i> migration of mesenchymal stem cells and wound healing in mice. In addition, we characterized cinnamtannin B-1-induced migration of mesenchymal stem cells pharmacologically and structurally. The mobilization of endogenous mesenchymal stem cells into the blood circulation was enhanced in cinnamtannin B-1-treated mice as shown by flow cytometric analysis of peripheral blood cells. Whole animal imaging analysis using luciferase-expressing mesenchymal stem cells as a tracer revealed that cinnamtannin B-1 increased the homing of mesenchymal stem cells to wounds and accelerated healing in a diabetic mouse model. Additionally, the cinnamtannin B-1-induced migration of mesenchymal stem cells was pharmacologically susceptible to inhibitors of phosphatidylinositol 3-kinase, phospholipase C, lipoxygenase, and purines. Furthermore, biflavonoids with similar structural features to cinnamtannin B-1 also augmented the migration of mesenchymal stem cells by similar pharmacological mechanisms. These results demonstrate that cinnamtannin B-1 promoted mesenchymal stem cell migration <i>in vivo</i> and improved wound healing in mice. Furthermore, the results reveal that cinnamtannin B-1-induced migration of mesenchymal stem cells may be mediated by specific signaling pathways, and the flavonoid skeleton may be relevant to its effects on mesenchymal stem cell migration.</p></div

Effects of various flavonoids and polyphenols on KUM6 cells.

<p>(A) Structure of cinnamtannin B-1 and MSC migration and proliferation after stimulation with cinnamtannin B-1, (B) procyanidin A2, (C) procyanidin B1, (D) morelloflavone, (E) cupressuflavone, (F) (+)-catechin, (G) genistein, (H) caffeic acid, and (I) gallic acid. Cell migration assay values are means ± SE and indices were normalized to platelet-derived growth factor (PDGF)-BB-treated cells; n = 4/group, ^*<i>P</i> < 0.05 and ^**<i>P</i> < 0.01 vs. control. Cell proliferation assay values are mean ± SE percentages normalized to that of untreated controls; n = 6/group, ^*<i>P</i> < 0.05 and ^**<i>P</i> < 0.01 vs. control.</p

Effects of cinnamtannin B-1 on migration of mouse mesenchymal stem cells (MSCs).

<p>(A) Increase in population of Lin−/PDGFRα+/Sca-1+ peripheral blood mononuclear cells of mice after systemic administration of cinnamtannin B-1, n = 6 per group. ^*<i>P</i> < 0.05 vs. control. (B) KUM6 cells localized in wound healing models; ffLuc-expressing KUM6 cells were injected 4 days after surgical incision (day 1). Images of a representative animal are shown on day 2 and 5 after ffLuc-KUM6 cell injection. (C) Quantification of photon counts at wound site on day 2 and 5 (n = 8–9 and 6–7, respectively); ^**<i>P</i> < 0.01 vs. control.</p