Streptomyces avermitilis is a gram-positive bacterium that undergoes complex physiological and morphological differentiation during its life cycle, which has implications in secondary metabolite production. Avermectin, produced by S. avermitilis, is widely used as an anthelmintic and insecticidal agent. In this study, we have applied Raman microspectroscopic imaging to elucidate the correlation between production of avermectin and the morphological differentiation in S. avermitilis. We demonstrate distinctive variations in the localization of secondary metabolites at various stages of morphological differentiation. Under solid culture, avermectin was detected in the mycelia formed at the later stages of morphological differentiation (e.g., spore-bearing mycelium and spiral spore chains), but not in the early-stage substrate mycelium. On the contrary, under liquid culture condition, avermectin was found concentrated in the mycelial pellet formed at the early MII stage of differentiation. Furthermore, the chemical profiles of the mycelia were substantially different depending on the culture condition. Raman spectra corresponding to proteins, lipids, and cytochrome were observed in the mycelia irrespective of the stage of morphological differentiation, however, carotenoid was observed under solid culture condition particularly in spore-bearing mycelium and spiral spore chains. KEY POINTS: • Avermectin production is regulated during mycelial differentiation • Liquid and solid culture conditions affects mycelial differentiation • Raman microspectroscopic analysis reveals localization profiles of avermectin.

The nitrogen rule in mass spectrometry was used to search for new nitrogen-compounds from microbial metabolites. During this program, two new nitrogen-containing compounds, penicidones E and F, were discovered from the filamentous fungal strain FKI-7498, which was isolated from soil collected in Tokushima, Japan and identified as Oidiodendron sp. by sequence analysis of the internal transcribed spacer region, including 5.8S ribosomal RNA. The structures of penicidones E and F were determined by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical modification analyses. These analyses revealed that penicidones E and F have a core structure of 3,5-dihydroxy-2-(4-pyridone-3-carbonyl)benzoic acid. Penicidone E exhibited hydroxyl radical scavenging activity.

Many useful natural products are usually screened based on their biological activities. On the other hand, various natural products can be detected based on their physicochemical properties. We have already reported the isolation and characterization of mangromicins from a cultural broth of Lechevalieria aerocolonigenes K10-0216 using physicochemical screening. In this report, we have conducted the mass spectrometry-based screening of new mangromicin analogs based on the neutral loss pattern originated from the unique cyclopentadecane skeleton of mangromicins. Two novel analogs were detected showing characteristic neutral loss pattern found in eight known mangromicin analogs. We propose the structures of the newly-found analogs based on the mass spectrometric as well as genomic and metabolic pathway data.

Sattahipmycin was isolated from the mycelium of marine-derived Streptomyces sp. GKU 257-1 by following the antibiofilm activity against E. coli NBRC 3972 throughout the purification steps. The structure of sattahipmycin was determined to be a new polycyclic xanthone related to xantholipin but lacking a dioxymethylene and a chlorinated carbon. This compound showed activity toward Gram-positive bacteria and Plasmodium falciparum, antibiofilm formation of Escherichia coli, and cytotoxicity to human cancer cell lines. Using genome sequence data, a biosynthetic pathway leading to sattahipmycin has been proposed involving an uncharacterized type II polyketide synthase biosynthetic gene cluster.

The mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest due to their diverse roles in biological systems and are far from being completely understood. Even though cellular cargo releases mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study, this aspect is investigated in detail using Raman microspectroscopic imaging. Considerable evidence is provided to suggest that the release of antibiotic penicillin by the filamentous fungus Penicillium chrysogenum involves EVs. Further, the study also reveals morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs. The results suggest a possible general role of EVs in the release of antibiotics from the producing organisms.

Trehangelins (THG) are newly identified trehalose compounds derived from broth cultures of an endophytic actinomycete, Polymorphospora rubra. THG are known to suppress Cellular Communication Network factor 1 (CCN1), which regulates collagen homeostasis in the dermis. Although the physical properties of THG suggest a high penetration of the stratum corneum, the effect of THG on the epidermis has not been reported. Here we describe a possible mechanism involved in skin aging focusing on the effect of THG on epidermal CCN1. This study shows that: (1) THG suppress epidermal CCN1 expression by inhibiting the translocation of Yes-Associated Protein (YAP) to nuclei. (2) Epidermal CCN1, localized at the basement membrane, regulates the balance between the growth and differentiation of keratinocytes. (3) Keratinocytes secrete more CCN1 than fibroblasts, which leads to disruption of the basement membrane and extracellular matrix components. (4) The secretion of CCN1 from keratinocytes is increased by ultraviolet B exposure, especially in aged keratinocytes, and deteriorates the elastic fiber structures in the underlying dermis. (5) Topical application of THG ameliorates the structure of the basement membrane in ex vivo human skin explants. Taken together, THG might be a promising treatment for aged skin by suppressing the aberrant YAP-CCN1 axis.

An actinomycete strain, LCR2-06T, isolated from a lichen sample on rock collected from Chiang Rai Province (Pong Phra Bat Waterfall), Thailand, was characterized using a polyphasic approach. The strain grew at 25-45 °C, pH 6-11 and on International Streptomyces Project 2 agar plate with 5 % (w/v) NaCl. It contained meso-diaminopimelic acid as the diamino acid in whole-cell hydrolysates. Rhamnose, ribose, xylose, madurose, glucose and galactose were detected as whole-cell sugar hydrolysates. Mycolic acids were absent. The N-acyl type of muramic acid was acetyl. The strain contained C16 : 0, TBSA 10-methyl C18 : 0 and 2-hydroxy C16 : 0 as the predominant fatty acids and MK-9(H6), MK-9(H4) and MK-9(H8) as the major menaquinones. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol and unidentified phospholipid. The draft genome of strain LCR2-06T was closely related to Actinomadura barringtoniae TBRC 7225T (99.2 %), Actinomadura nitritigenes NBRC 15918T (98.8 %), Actinomadura montaniterrae TISTR 2400T (98.5 %) and Actinomadura physcomitrii JCM 33455T (97.9 %). The draft genome of LCR2-06T was 11.1 Mb with 10 588 coding sequences with an average G+C content of 72.7 mol%. Results of genomic analysis revealed that the ANIb and ANIm values between strain LCR2-06T and A. montaniterrae TISTR 2400T were 90.0 and 92.0 %, respectively. The digital DNA-DNA hybridization value was 43.9 % in comparison with the draft genome of A. montaniterrae TISTR 2400T. The strain produced an antibacterial compound active against Bacillus subtilis ATCC 6633 and Kocuria rhizophila ATCC 9341. The results of taxonomic analysis suggested that strain LCR2-06T represented a novel species of the genus Actinomadura for which the name Actinomadura violacea sp. nov. is proposed. The type strain is LCR2-06T (=JCM 33065T=KCTC 49547T=NBRC 114810T=LMG 32136T=TISTR 2935T).

Nanaomycin K, derived from Streptomyces rosa subsp. notoensis OS-3966T, has been discovered to have inhibitory bioactivity on epithelial-mesenchymal transition (EMT), an important mechanism of cancer cell invasion and migration. In this study, we examined the anti-EMT and anti-tumor effect of nanaomycin K in bladder cancer, where EMT has important roles in progression. We treated two bladder cancer lines, non-muscle-invasive KK47 and muscle-invasive T24, with nanaomycin K to determine the effects on cell proliferation, apoptosis and expression of EMT markers in vitro. Wound-healing assays were performed to assess cell invasion and migration. We conducted an in vivo xenograft study in which mice were inoculated with bladder cancer cells and treated with intratumoral administration of nanaomycin K to investigate its anti-tumor and EMT inhibition effects. As the results, nanaomycin K (50 µg/mL) significantly inhibited cell proliferation in KK47 (p < 0.01) and T24 (p < 0.01) in the presence of TGF-β, which is an EMT-inducer. Nanaomycin K (50 µg/mL) also significantly inhibited cell migration in KK47 (p < 0.01) and T24 (p < 0.01), and induced apoptosis in both cell lines in the presence of TGF-β (p < 0.01). Nanaomycin K increased the expression of E-cadherin and inhibited the expression of N-cadherin and vimentin in both cell lines. Nanaomycin K also decreased expression of Snail, Slug, phospho-p38 and phospho-SAPK/JNK especially in T24. Intratumoral administration of nanaomycin K significantly inhibited tumor growth in both KK47 and T24 cells at high dose (1.0 mg/body) (p = 0.009 and p = 0.003, respectively) with no obvious adverse events. In addition, nanaomycin K reversed EMT and significantly inhibited the expression of Ki-67 especially in T24. In conclusion, we demonstrated that nanaomycin K had significant anti-EMT and anti-tumor effects in bladder cancer cells, suggesting that nanaomycin K may be a therapeutic candidate for bladder cancer treatment.

An anti-mannheimiosis agent, aldsulfin, was isolated from a culture broth of the fungus Lasiodiplodia pseudotheobromae FKI-4499, together with a known compound, lasiodipline C, using bioassay-guided fractionation. Spectroscopic analysis of aldsulfin, using NMR, mass spectrometry, and CD analyses revealed it to be an epithiodiketopiperazine with an unstable and unusual hemithioaminal moiety. Aldsulfin showed antibacterial activity against Mannheimia haemolytica and Pasteurella multocida.

Raman spectra are molecular structure-specific and hence are employed in applications requiring chemical identification. The advent of efficient handheld and smartphone-based Raman instruments is promoting widespread applications of the technique, which often involve less trained end users. Software modules that enable spectral library searches based on spectral pattern matching is an essential part of such applications. The Raman spectrum recorded by end users will naturally have varying levels of signal to noise (SN), baseline fluctuations, etc., depending on the sample environment. Further, in biological, forensic, food, pharmaceuticals, etc., fields where a vast amount of Raman spectral data is generated, careful removal of background is often impossible. In other words, a 100% match between the library spectrum and user input cannot be often guaranteed or expected. Often, such influences are discounted upon developing mathematical methods for general applications. In this manuscript, we carefully examine how such effects would determine the results of spectral similarity-based library search. We show that several popular mathematical spectral matching approaches give incorrect results under the influence of small changes in the baseline and/or the noise. We also discuss the points to be carefully considered while generating a spectral library. We believe our results will be a guiding note for developing applications of Raman spectroscopy that uses a standard spectral library and mathematical spectral matching.

Raman microspectroscopy is a minimally invasive technique that can identify molecules without labeling. In this study, we demonstrate the detection of penicillin G inside Penicillium chrysogenum KF425 fungal cells. Raman spectra acquired from the fungal cells had highly overlapped spectroscopic signatures and hence were analyzed with multivariate curve resolution by alternating least-squares (MCR-ALS) to extract the spectra of individual molecular constituents. In addition to detecting spatial distribution of multiple constituents such as proteins and lipids inside the fungal body, we could also observe the subcellular localization of penicillin G. This methodology has the potential to be employed in screening the production of bioactive compounds by microorganisms.

Two new nitrogen-containing metabolites, designated hatsusamide A (1) and B (2), were isolated from a culture broth of Penicilliumsteckii FKJ-0213 together with the known compounds tanzawaic acid B (3) and trichodermamide C (4) by physicochemical (PC) screening. The structures of 1 and 2 were determined as a tanzawaic acid B-trichodermamide C hybrid structure and a new analog of aspergillazines, respectively. The absolute configuration of 1 was determined by comparing the values of tanzawaic acid B and trichodermamide C in the literatures, such as 1H-nuclear magnetic resonance (1H-NMR) data and optical rotation, after hydrolysis of 1. Compounds 1-4 were evaluated for cytotoxicity and anti-malarial activities. Compounds 1 and 3 exhibited weak anti-malarial activity at half-maximal inhibitory concentration (IC50) values of 27.2 and 78.5 µM against the K1 strain, and 27.9 and 79.2 µM against the FCR3 strain of Plasmodium falciparum, respectively. Furthermore, 1 exhibited cytotoxicity against HeLa S3, A549, Panc1, HT29 and H1299 cells, with IC50 values of 15.0, 13.7, 12.9, 6.8, and 18.7 μM, respectively.

A new chaetochiversin analog, designated chaetochiversin C (1), was discovered from a cultured broth of fungal strain FKI-7792 by physicochemical screening. This strain was identified as a member of genus Neocosmospora based on morphology and DNA barcoding. The partially relative configuration of 1 was determined by 13C-NMR chemical shifts of the acetonide analog of 1. The absolute configuration was determined using an advanced Mosher's method. Compound 1 was assessed for anti-tumor, anti-microbial, and anti-malarial activities, and its ability to scavenge or quench reactive oxygen species (ROS), such as superoxide anion radicals, hydroxy radicals and singlet oxygen (1O2). Compound 1 showed a quenching effect on 1O2.

The present study investigated whether or not the oral administration of trehangelin-A (THG-A) is effective for metabolic disorders caused by a high-fat diet, as we previously showed that the intraperitoneal administration of THG-A improved metabolic disorders caused by a high-fat diet. Mice received a con-trol diet or high-fat diet for eight weeks. Concurrently, mice were orally administered 0.2 ml/mouse phosphate-buffered saline (PBS) or 1 or 10 mg/0.2 ml/mouse of THG-A once daily during the experiment. The weight gain caused by a high-fat diet was significantly suppressed by oral THG-A compared to a high-fat diet without THG-A. In addition, at eight weeks after starting the diet, the increased plasma total-cholesterol (T-CHO) and low-density lipoprotein-cholesterol (LDL-C) levels caused by a high-fat diet were significantly reduced by 10 mg/mouse THG-A and tended to attenuated by 1 mg/mouse THG-A. The LDL receptor and CYP7A1 mRNA expression in liver associated with lipid metabolism for reducing plasma LDL-C levels was significantly enhanced by oral THG-A. In contrast, oral THG-A exerted no marked effects on mice fed the control diet. The dysbiosis of a high-fat diet fed mice, which is in the form of an increased Firmicutes-to-Bacteroidetes ratio, also recovered, and the high-fat diet induced decreased levels of Bacteroides and Akkermansia genera, which are beneficial microbiota against metabolic disorders, were also restored by oral THG-A. These results indicate that oral THG-A administration acts on metabolic disorders by improving the lipid metabolism and restoring beneficial microbiota to resolve high-fat diet induced dysbiosis. (C) 2020 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

Two new sulfur compounds, designated thioporidiol A (1) and B (2), were discovered by the MoS-screening program from a culture broth of Trichodermapolypori FKI-7382. The structures of 1 and 2 were determined as C13 lipid structures with an N-acetylcysteine moiety. The relative configuration at the C-5 and C-6 position of 1 was determined by the derivatives of -methoxy--phenylacetic acid diesters, and the absolute configuration of the N-acetylcysteine moiety was determined by advanced Marfey's analysis. Compounds 1 and 2 were evaluated for anti-microbial, cytotoxic and anti-malarial activities. Compound 2 exhibited anti-microbial activity against Candida albicans ATCC 64548.

Trichoderma virens FKI-7573の代謝産物から、奇数の窒素原子を持つ化合物であることを示す奇数分子量ガイド質量分析法を用いて、新規窒素含有化合物のtrichothioneic acid(TA)を見出した。FKI-7573株は北海道帯広市で採取された土壌から単離され、5.8SリボソームRNAを含むITS領域の配列分析により、Trichoderma virensと同定された。TAの構造を質量分析、核磁気共鳴、電子円二色スペクトルと化学分解分析により決定した。その結果、TAはヘプテリジン酸とL-エルゴチオネインから成っており、3つの窒素原子を含んでいた。TCAはヒドロキシルラジカル除去活性とsinglet oxygen消去活性を示した。

新規ナナオマイシン類似体のナナオマイシンKを、放線菌株"Streptomyces rosa subsp. notoensis"OS-3966の培養液から単離した。核磁気共鳴(NMR)分析により、ナナオマイシンKの平面構造にエルゴチオネイン部分が認められた。絶対配置を決定するために、ナナオマイシンKをナナオマイシンEとL-エルゴチオネインの標準品を用いて半合成した。天然および半合成ナナオマイシンKは、保持時間、質量分析、1HNMRおよび旋光度データに基づいて、同じ化合物として同定された。ナナオマイシンKは、トランスフォーミング増殖因子β1により誘発される上皮間葉移行を起こしているMadin-Darbyイヌ腎臓細胞に対して細胞毒性を示した。

Penicillium brevicompactum FKJ-0123の培養ブロスから、物理化学的スクリーニングにより、新規のN-シンナモイルトリペプチドcipralphelin(I)を単離した。Iの精製にはシリカゲルおよびODSカラムによるクロマトグラフィーを用い、次いで合成的HPLC法を用いた。Iの構造決定には1次元/2次元NMRスペクトル解析法と高分解能質量分析法を中心とした分光分析法を用い、IがN-シンナモイル-プロピル-アラニル-フェニルアラニン・メチルエステルであった。Iを構成する3種類のアミノ酸の絶対構造は、その加水分解物にMarfey法を適用して決定した。Iの細胞毒性、抗菌活性およびスーパーオキシドアニオンラジカルなどの活性酸素種の捕捉能を評価し、Iはヒドロキシラジカルに対して強力な捕捉能を有していた。

多剤感受性Saccharomyces cerevisiae 12geneΔ0HSR-iERG6株を用いたバイオアッセイ誘導分画により、Fusarium sp. FKI-7550の培養ブロスから新規化合物fusaramin(I)と共に3種類の既知化合物サンブトキシンとその類似体(II〜IV)を単離した。Iの絶対構造は1次元/2次元NMRスペクトル解析法と円偏光二色性で決定した。Iは数種のグラム陽性/陰性菌に対する抗菌活性を示し、グリセロール含有培地で生育するS.cerevisiae 12geneΔ0HSR-iERG6の増殖を阻害した。グリセロール含有培地で生育する野生型と多剤感受性型の上記酵母に対するMIC値はそれぞれ>128と0.64μg/mLであったが、グルコース含有培地で生育する上記酵母に対するMIC値は>128μg/mLであった。化合物I〜IVはいずれも、単離したS.cerevisiaeミトコンドリアを用いた酸化的リン酸化を経由してATP合成を阻害した。

Whole genome analysis of Streptomyces sp. KO-7888 has revealed various pathway-specific transcriptional regulatory genes associated with silent biosynthetic gene clusters. A Streptomyces antibiotic regulatory protein gene, speR, located adjacent to a novel nonribosomal peptide synthetase (NRPS) gene cluster, was overexpressed in the wild-type strain. The resulting recombinant strain of Streptomyces sp. KO-7888 produced two new lipopeptides, sarpeptins A and B. Their structures were elucidated by high-resolution electrospray ionization mass spectrometry, NMR analysis, and the advanced Marfey's method. The distinct modular sections of the corresponding NRPS biosynthetic gene cluster were characterized, and the assembly line for production of the lipopeptide chain was proposed.

The purpose of this study is to determine whether or not trehangelin A (THG-A) is effective in treating the metabolic clinical condition caused by a high-fat diet. The body weight, epididymal adipose volume, alanine transaminase (ALT), total-cholesterol (T-CHO), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and glucose concentrations in serum increased in mice fed a high-fat diet compared to mice fed a control diet. On the other hand, adiponectin level in serum of mice fed a high-fat diet decreased compared to that of control mice. When mice fed a high-fat diet were intraperitoneally administered THG-A of 20 mg/kg three times per week, the levels of TG and glucose in serum were significantly reduced compared to those fed high-fat without THG-A. Interestingly, the levels of high-density lipoprotein cholesterol (HDL-C) in serum were increased by THG-A administration in both mice fed a control diet and those fed high-fat diet. The decreased level of adiponectin by a high-fat diet was also recovered by THG-A treatment. The liver expression of mRNA from pro-inflammatory cytokines, including interleukin (IL)-6 and tumor necrosis factor (TNF)-α, were significantly increased in mice fed a high-fat diet compared to those fed a control diet. However, the increased IL-6 levels in mice fed a high-fat diet were significantly suppressed by THG-A treatment. Furthermore, the increased expression of TNF-α mRNA or COL1A2 mRNA by a high-fat diets tended to be decreased in mice treated with THG-A. These results show that THG-A treatment attenuates the progression of metabolic clinical conditions, suggesting its potential efficacy against obesity-related metabolic disorders.

Two new antibiotics, designated virantmycin B (1) and C (2), were isolated from the cultured broth of Streptomyces sp. AM-2504. Compounds 1 and 2 were purified by Diaion HP-20, silica gel, and octadecylsilane chromatography, followed by high-performance liquid chromatography. The chemical structures of the new compounds, 1 and 2, were determined by nuclear magnetic resonance and mass spectrometry, as containing a terahydroquinoline and an indoline, respectively, each also containing a hydroxy cyclopentenone moiety. Both compounds demonstrated weak antimicrobial (both antibacterial and antifungal) activity and compound 1 also showed antiviral activity against the dengue virus, whereas compound 2 exhibited no antiviral properties.

Two new nanaomycin analogs, nanaomycin I and J, were isolated from a cultured broth of an actinomycete strain, "Streptomyces rosa subsp. notoensis" OS-3966. In our previous study, we have confirmed the occurrence of nanaomycin I (m/z = 482 [M + H]+) that lacks a pseudo-disaccharide from the mycothiol of nanaomycin H under same culture condition. In this study, to confirm the structure of nanaomycin I, the strain "S. rosa subsp. notoensis" OS-3966 was re-cultured and the target compound with m/z = 482 [M + H]+ was isolated. Furthermore, we discovered another new analog, designated as nanaomycin J in isolating nanaomycin I. The NMR analyses revealed that the structures of nanaomycin I and J are N-acetylcysteine S-conjugates without a pseudo-disaccharide and N-acetylcysteine S-conjugates without a myo-inositol of nanaomycin H, respectively. The relative configurations of the tetrahydropyrane moiety of nanaomycin I and J were determined by rotating-frame overhauser effect spectroscopy (ROESY) analysis. Absolute configurations of the N-acetylcysteine moiety of nanaomycin I and J were determined by advanced Marfey's analyses for acid hydrolysis of de-sulfurized nanaomycin I and J with Raney nickel. Nanaomycin I and J showed moderate cytotoxicity against several human tumor cell lines.

Epithelial-mesenchymal transition (EMT), phenotypic changes in cell adhesion and migration, is involved in cancer invasion and metastasis, hence becoming a target for anti-cancer drugs. In this study, we report a method for the evaluation of EMT inhibitors by using a photoactivatable gold substrate, which changes from non-cell-adhesive to cell-adhesive in response to light. The method is based on the geometrical confinement of cell clusters and the subsequent migration induction by controlled photoirradiation of the substrate. As a proof-of-concept experiment, a known EMT inhibitor was successfully evaluated in terms of the changes in cluster area or leader cell appearance, in response to biochemically and mechanically induced EMT. Furthermore, an application of the present method for microbial secondary metabolites identified nanaomycin H as an EMT inhibitor, potentially killing EMTed cells in disseminated conditions. These results demonstrate the potential of the present method for screening new EMT inhibitors.

New natural products, designated pochoniolides A and B, were isolated from the cultured broth of fungal strain FKI-7537 using a physicochemical screening methodology. Strain FKI-7537 was isolated from a soil sample collected at Niijima, Tokyo, Japan and identified as Pochonia chlamydosporia var. spinulospora by morphological characteristics and DNA sequence analysis. The chemical structures of pochoniolides A and B were elucidated by NMR and mass spectra and found to be new compounds consisting of a muconolactone moiety connected with a chromone unit. Pochoniolides A and B were identified as racemate mixtures using data on optical rotation and circular dichroism spectra. Furthermore, enantiomers of pochoniolide B, pochoniolides B1 and B2, were separated using a chiral HPLC column. Pochoniolides A and B showed hydroxyl radical-scavenging and singlet oxygen-quenching activities.

1. Changes in Main Body Paragraphs There are some mistakes in this article [1]. Ref. 8 in line 47 of page 2 should be ref. 9
ref. 9 in line 6 of page 3 should be ref. 10
ref. 10 in line 9 of page 3 should be ref.11
ref. 11 in line 13 of page 3 should be deleted
ref. 12–14 in line 24 of page 3 should be ref. 12,13
ref. 15 in line 26 of page 3 should be ref. 14
ref. 16 in line 34 of page 3 should be ref. 15,16
“1981” in line 4 of Section 4 should be “1971”. 1. Original: The actinomycetes, particularly species from the genus Streptomyces, have proved to be a tremendous high-impact source of valuable chemicals. They have yielded many clinically essential antimicrobial compounds, including streptomycin, actinomycin, and streptothricin [8]. Revised: The actinomycetes, particularly species from the genus Streptomyces, have proved to be a tremendous high-impact source of valuable chemicals. They have yielded many clinically essential antimicrobial compounds, including streptomycin, actinomycin, and streptothricin [9]. 2. Original: Incidentally, salinosporamide A, which holds promise for development of an anticancer drug, is produced by a strain of the genus Salinispora, a rare actinomycete isolated from a heat-treated marine sediment sample [9]. Revised: Incidentally, salinosporamide A, which holds promise for development of an anticancer drug, is produced by a strain of the genus Salinispora, a rare actinomycete isolated from a heat-treated marine sediment sample [10]. 3. Original: It is believed that the actinomycetes are the source of some 61% of all microorganism-derived bioactive substances so far discovered [10]. Revised: It is believed that the actinomycetes are the source of some 61% of all microorganism-derived bioactive substances so far discovered [11]. 4. Original: This suggests that rare actinomycetes are a valuable source of novel compounds, and that improved isolation strategies are required to increase the frequency in which they are isolated [11]. Revised: This suggests that rare actinomycetes are a valuable source of novel compounds, and that improved isolation strategies are required to increase the frequency in which they are isolated. 5. Original: Decades of success in our exploration of the actinomycetes is exemplified by the discovery in the Kitasato Institute by Satoshi Ōmura in the early-1970s of Streptomyces avermectinius (synonym S. avermitilis) MA-4680T, the microbe which produces the avermectins [12–14]. Revised: Decades of success in our exploration of the actinomycetes is exemplified by the discovery in the Kitasato Institute by Satoshi Ōmura in the early-1970s of Streptomyces avermectinius (synonym S. avermitilis) MA-4680T, the microbe which produces the avermectins [12,13]. 6. Original: The (ivermectin derivative, ivermectin, is perhaps the world's greatest, most effective, and safest drug for the treatment and prevention of a diverse range of human diseases and conditions [15]. Revised: The avermectin derivative, ivermectin, is perhaps the world's greatest, most effective, and safest drug for the treatment and prevention of a diverse range of human diseases and conditions [14]. 7. Original: The award citation stated "William C. Campbell and Satoshi Omura discovered a new drug, avermectin, the derivatives of which have radically lowered the incidence of River Blindness and Lymphatic Filariasis, as well as showing efficacy against an expanding number of other parasitic diseases" [16]. Revised: The award citation stalted "William C. Campbell and Satoshi Omura discovered a new drug, avermectin, the derivatives of which have radically lowered the incidence of River Blindness and Lymphatic Filariasis, as well as showing efficacy against an expanding number of other parasitic diseases" [15,16]. 8. Original: KML strain OS-3601 (Figure 1) was isolated from a soil sample collected at Aso, Kumamoto prefecture, Japan, in 1981, Revised: KML strain OS-3601 (Figure 1) was isolated from a soil sample collected at Aso, Kumamoto prefecture, Japan, in 1971,.

In the present study, we evaluated the prooxidative mode of action of photoirradiated (+)-catechin at 400 nm in relation to reactive oxygen species generation and its possible application to disinfection. Photoirradiation of (+)-catechin at a concentration of 1 mg/mL yielded not only hydrogen peroxide (H2O2) but hydroxyl radical (·OH) in a total amount of approximately 20 μM in 10 min. As a result, photoirradiated catechin killed Staphylococcus aureus, and a > 5-log reduction in viable bacteria counts was observed within 20 min. Liquid chromatography-high-resolution-electrospray ionization-mass spectrometry showed that photoirradiation decreased the (+)-catechin peak (molecular formula C15H14O6) whilst it increased two peaks of a substance with the molecular formula C15H12O6 with increasing irradiation time. Nuclear magnetic resonance analysis revealed that the two C15H12O6 peaks were allocated to intramolecular cyclization products that are enantiomers of each other. These results suggest that photoirradiation induces oxidation of (+)-catechin resulting in the reduction of oxygen to generate H2O2. This H2O2 is then homolytically cleaved to ·OH, and alongside this process, (+)-catechin is finally converted to two intramolecular cyclization products that are different from the quinone structure of the B ring, as proposed previously for the autoxidation and enzymatic oxidation of catechins.

The multidrug-sensitive budding yeast, Saccharomyces cerevisiae 12geneΔ0HSR-iERG6, is very useful in antifungal screens. A novel compound, named pestynol (1), was discovered from a culture of the fungus Pestalotiopsis humus FKI-7473 using the multidrug-sensitive yeast. The structure of 1 was elucidated by NMR studies and modified Mosher's method as (1 R,2 R,3 R,4 R)-( E)-5-(7,11-dimethyl-3-methylenedodeca-6,10-dien-1-yn-1-yl)cyclohex-5-ene-1,2,3,4-tetraol. Compound 1 showed antimicrobial activity against the Gram-positive bacteria, Klebsiella pneumoniae, and S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus, but displayed only weak cytotoxicity against various human cancer cell lines. Compound 1 displayed antifungal activities against S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus at 10 μg/disc.

Global public health faces a desperate situation, due to the lack of effective antibiotics. Coordinated steps need to be taken, worldwide, to rectify this situation and protect the advances in modern medicine made over the last 100 years. Work at Japan's Kitasato Institute has been in the vanguard of many such advances, and work is being proactively tailored to promote the discovery of urgently needed antimicrobials. Efforts are being concentrated on actinomycetes, the proven source of most modern antibiotics. We devised a novel physicochemical screening mechanism, whereby simple physico-chemical properties, in conjunction with related detection methods, such as LC/MS, LC/UV, and polarity, could be used to identify or predict new compounds in a culture broth, simply by comparing results with existing databases. New compounds are isolated, purified, and their structure determined before being tested for any bioactivity. We used lyophilized actinomycete strains from the Kitasato Microbial Library, most more than 35 years old, and found 330 strains were producers of useful bioactive substances. We also tested organisms found in fresh samples collected in the complex environments from around plant roots, as well as from sediments of mangrove forests and oceans, resulting in the discovery of 36 novel compounds from 11 actinomycete strains. A compound, designated iminimycin, containing an iminium ion in the structure was discovered from the culture broth of Streptomyces griseus OS-3601, which had been stored for a long time as a streptomycin-producing strain. This represented the first iminium ion discovery in actinomycetes. Compounds with a cyclopentadecane skeleton containing 5,6-dihydro-4-hydroxyl-2-pyrone ring and tetrahydrofuran ring, designated mangromicins, were isolated from the culture broth of Lechevalieria aerocolonigenes K10-0216 obtained from sediment in a mangrove forest. These structures are extremely unique among natural compounds. From the same culture broth, new steroid compounds, named K10-0216 KA and KB, and other new compounds having a thiazole and a pyridine ring, named pyrizomicin A and B, were discovered. New substances can be found from actinomycetes that have been exhaustively studied. Novel compounds with different skeletons can be found from a single broth of one strain. The sought after new antibiotics will arise from continued exploitation of the actinomycetes, especially rare actinomycetes. Work on new organisms and samples should be augmented by re-examination of known actinomycetes already in storage. New research should also be carried out on the manipulation of culture media, thereby stimulating actinomycete strains to produce novel chemicals. The establishment of wide-ranging international research collaborations will facilitate and expedite the efficient and timely discovery and provision of bioactive compounds to help maintain and promote advances in global public health.

Fungal strain FKJ-0025 was isolated from deep-sea sediment collected at the Wakamiko Caldera in Kagoshima Bay (water depth: 200 m). The fungal strain FKJ-0025 was identified as the genus Sarcopodium based on its morphology and internal transcribed spacer (ITS) sequence. Two new compounds, designated sarcopodinols A (1) and B (2), were isolated together with the known compound SF-227 (3).

Two new compounds, designated as hamuramicins A (1) and B (2), were isolated from the cultured broth of an endophytic actinomycete Allostreptomyces sp. K12-0794 by silica gel column chromatography and HPLC. The structures of 1 and 2 were elucidated as 22-membered macrolide containing triene and trienone with an alkyl side chain by spectroscopic analyses including NMR experiments. Both compounds showed growth inhibition activity against Kocuria rhizophia and Xanthomonas oryzae pv. oryzae as well as human cell line toxicity.

Actinoallolides are anti-trypanosomal macrolides isolated from the secondary metabolites of two endophytic actinomycete strains, Actinoallomurus fulvus MK10-036 and K09-0307. A putative actinoallolide biosynthetic gene cluster was predicted from the genome sequence of the strain K09-0307. The gene cluster spans a contiguous 53 kb DNA region that comprises seven genes encoding three PKSs (aalA1, aalA2, and aalA3), cytochrome P450 (aalB), acyl-CoA dehydrogenase (aalC), crotonyl-CoA reductase (aalD), and TetR family regulator (aalR). The entire gene cluster was cloned into a plasmid pYIK1 by assembling DNA fragments, which were obtained from two cosmids containing left and right parts of the gene cluster. Following the introduction of an ermE* promoter at 100bp upstream from the start codon of aalA1, the gene cluster was introduced into Streptomyces coelicolor M1152. Subsequent LC-MS analysis revealed production of actinoallolide A in the culture broth. Thus, the actinoallolide biosynthetic gene cluster was identified by heterologous expression in Streptomyces.

Two new antibiotics, designated pyrizomicin A and B, were isolated from the cultured broth of a rare actinomycete strain, Lechevalieria aerocolonigenes K10-0216, by silica gel and HPLC purification. The chemical structures of pyrizomicin A and B were elucidated as new thiazolyl pyridine compounds by nuclear magnetic resonance and mass spectrometry. Pyrizomicin A and B both showed antimicrobial activity.

In a search for compounds interacting with ergosterol resin, a new compound named dipyrimicin B was isolated from a rare actinomycete strain, Amycolatopsis sp. K16-0194. In addition, another analog, dipyrimicin A, which does not interact with the resin, was also discovered. The structures of the two dipyrimicins were established by comprehensive 1D and 2D NMR and MS analyses and found to contain a unique core structure, a 2,2'-bipyridine skeleton. Dipyrimicin A showed strong antimicrobial and cytotoxic activity, whereas dipyrimicin B displayed distinctly poor antimicrobial and cytotoxic activities.

A new antibiotic, designated mumiamicin, was isolated from the cultured broth of the rare actinomycete strain, Mumia sp. YSP-2-79, by Diaion HP-20, silica gel and ODS column chromatography, followed by HPLC purification. The chemical structure of mumiamicin was elucidated as a new furan fatty acid by nuclear magnetic resonance and mass spectrometry. Mumiamicin showed antimicrobial activity and antioxidative activity.

Jietacins, an azoxy antibiotic class of chemicals, were isolated from the culture broth of Streptomyces sp. KP-197. They have a unique structural motif, including a vinyl azoxy group and a long acyclic aliphatic chain, which is usually branched but non-branched in the case of jietacin C. During a drug discovery program, we found that jietacins display potent anthelmintic activity against parasitic nematodes and that jietacin A has a moderate or low acute toxicity (LD50 > 300 mg/kg) and no mutagenic potential in a mini Ames screen. This suggests that jietacins have potential for drug discovery research. In order to create a novel anthelmintic agent, we performed design, synthesis, and biological evaluation of jietacin derivatives against parasitic nematodes. Of these derivatives, we found that a fully synthesized simplified derivative exhibited better anthelmintic activity against three parasitic nematodes than natural jietacins. In addition, it had a better efficacy in vivo through oral administration against a mouse nematode. This indicated that the azoxy motif could prove useful as a template for anthelmintic discovery, possibly creating a class of anthelmintic with novel skeletons, a potential new mode of action, and providing further insight for rational drug design.

Bisoxazolomycin (1), oxazolomycin A2 (2) and oxazolomycin A (3) were identified by physicochemical screening approach from a culture broth of 'Streptomyces subflavus subsp. irumaensis' AM-3603. Compound 2 is a hydrolyzed analog of 3 at the β-lactone ring, and 1 is a new dimeric analog of 2. Compounds 1 and 2 exhibited less potent antibacterial activity and cytotoxicity than 3, which might be due to lack of the β-lactone ring.

This corrects the article DOI: 10.1038/ja.2015.142.

The Ōmura research group of the Kitasato Institute has isolated multiple microorganisms over a period of five decades. The resulting collection comprises a broad spectrum of microbes, including strains producing novel and diverse compounds with biological activities. A bioassay-guided fractionation of microbial culture broths has been employed to screen the microbial collection for compounds with new biological activities. And numerous novel natural products have been discovered among the microbial metabolites produced by members of the collection. However, dereplication of already known compounds and their potential analogs is a vital part of the discovery process of new microbial natural products. Recently, it has become easy to acquire the ultraviolet (UV) and mass spectrometry (MS) spectra of many single components of microbial culture broths in combination with high-performance liquid chromatography. To achieve most effective utilization of our microbial library, new compounds from microbial culture broths were investigated by employing an approach based on the physico-chemical properties using spectral analyses such as UV and MS and color reaction, collectively designated as physicochemical (PC) screening. As a result of physicochemical screening, many new compounds were identified among the secondary metabolites of fresh isolated rare actinomycetes and Streptomyces spp. preserved for a long time as producer of biological compounds. In this review, we introduce the Kitasato microbial library and the new compounds discovered from the library by PC screening.

Physicochemical screening identified a new nanaomycin analog, nanaomycin H, which was isolated from a culture broth of Streptomyces rosa subsp. notoensis OS-3966. This microorganism is already known to produce seven nanaomycin compounds, (nanaomycin A to G). Structural elucidation of nanaomycin H showed it to be a pyranonaphthoquinone with a mycothiol moiety. A N-acetylcysteine S-conjugate of nanaomycin H, without α-glucosamine linked to myo-inositol moiety, mercapturic acid derivative, was also detected in the same culture broth. Mercapturic acid derivatives of secondary metabolites are known to be produced for xenobiotic metabolism outside microbial cells. Mycothiol acts as a detoxifier to help prevent cell damage from factors such as oxidative stress. The production of O2- generated by reduction of nanaomycin A is correlated with antibacterial activity. Mycothiol-containing nanaomycin H proved to be markedly decreased in O2- and did not express any notable antimicrobial activity. It is suggested that nanaomycin H is produced in the detoxification process.

A new decalin, decatamariic acid, was isolated from a cultured broth of the fungus Aspergillus tamarii FKI-6817. Its absolute configuration was elucidated by NMR and electronic circular dichroism. Decatamariic acid (10 μM) elicited ~50% inhibition of the ATP production in mitochondria isolated from wild-type Saccharomyces cerevisiae without affecting the activities of respiratory enzymes. The action manner of this compound may be interesting as a possible seed for new pesticides.

Pueraria candollei Grah. ex Benth. is a Thai herb that contributes to many phytoestrogenic-related pharmacological activities. In this study, the diversity of endophytic actinomycetes from roots and root nodules of P. candollei and actinomycetes from rhizospheric soils were investigated. Moreover, the secondary metabolites production of these isolated actinomycetes was investigated. A total of 85 isolates were obtained from roots, root nodules and rhizospheric soils, 32 from the roots of P. candollei var. candollei, 26 from the root nodules of P. candollei var. mirifica and 27 from rhizospheric soil samples. Partial 16S rRNA sequence data revealed that the isolated actinomycetes were distributed among 7 genera including, Streptomyces, Micromonospora, Verrucosispora, Microbispora, Nonomuraea, Plantactinospora and Shimazuella. The majority of isolates from plant sources were members of the genus Micromonospora. Broth extracts of these isolates were analyzed using LC/UV and LC/MS. The physicochemical properties of 10 selected peaks were searched in the database. The physicochemical properties of some peaks were matched with known microbial metabolites. The structure of an unknown compound was elucidated using spectroscopic data. The NMR data indicated the presence of N-acetylhomocysteine moiety and the compound was determined to be S-adenosyl-N-acetylhomocysteine. This compound was isolated from Micromonospora for the first time. In conclusion, this work contributed to the known information on the diversity of actinomycetes associated with P. candollei and their capability for secondary metabolite production.

An enantioselective total synthesis of (+)-mangromicin A has been accomplished. The tetrahydrofuran ring of mangromicin A, possessing a tetrasubstituted carbon center, was constructed by Mukaiyama-type vinylogous alkylation via a cyclic oxocarbenium intermediate derived from a γ-hydroxy ketone with ideal stereoselectivity, and the 4-hydroxydihydropyrone scaffold was generated via Dieckmann cyclization at a late stage of the total synthesis. The reliable asymmetric synthesis of (+)-mangromicin A has revealed the absolute configuration of naturally occurring mangromicin A.

A new antibiotic, designated sagamilactam, was isolated from the cultured broth of Actinomadura sp. K13-0306 by Diaion HP-20, silica gel and octadecylsilane column chromatography followed by purification by HPLC. The chemical structure of the new compound was elucidated by spectroscopic analyses, including NMR and MS. The structure of sagamilactam proved to be a new compound consisting of a polyunsaturated and polyoxygenated 34-membered macrocyclic lactam containing diene, triene and tetraene conjugated olefins and a decalin moiety. Sagamilactam showed antitrypanosomal activity with an IC50 value of 0.25±0.11 μM.

It is the major characteristic of the cell-wall peptidoglycan structure in members of the family Micromonosporaceae that N-acetylmuramic acid (MurNAc) of glycan strand is replaced with N-glycolylmuramic acid (MurNGlyc). Consequently, it is difficult to use enzymatic methods for their peptidoglycan analyses. We therefore developed analysis method of peptidoglycan without using cell wall lytic enzymes as example to take the 3 genera, Micromonospora, Catenuloplanes, and Couchioplanes belonging to the family Micromonosporaceae, and their peptidoglycans were partially hydrolyzed with 4 M HCl at 60°C for 16 h followed by derivatization with N(α)-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide (FDLA) or 1-phenyl-3-methyl-5-pyrazolone (PMP) and LC/MS analysis. Peptidoglycan of the genus Micromonospora consisted of a MurNGlyc-Gly-D-Glu-meso-diaminopimelyl (DAP)-D-Ala peptide stem and direct linkage between D-Ala and meso-DAP. In contrast, peptidoglycans of the genera Catenuloplanes and Couchioplanes consisted of a MurNGlyc-Gly-D-Glu-L-Lys-D-Ala peptide stem, and cross-linkage between D-Ala and L-Lys was mediated by an L-Ser residue. This method can be used to analyze the cell-wall peptidoglycan structure of other bacteria as well. By derivatization with FDLA or PMP followed by LC/MS analysis, the structure can be determined using only 0.2 mg of purified peptidoglycan.

Trehangelins are trehalose angelates discovered from endophytic actinomycete Polymorphospora rubra K07-0510. We identified the trehangelin biosynthetic gene cluster, including genes that encode a glycoside hydrolase-like protein (thgC), α-amylase (thgD), 3-ketoacyl-ACP synthase III (thgI), 3-ketoacyl-ACP reductase (thgK), enoyl-CoA hydratase (thgH) and acyl transferase (thgJ). Heterologous expression of thgH, thgI, thgJ and thgK confirmed the importance of these genes in the biosynthesis of trehangelin A. Enzymatic activity studies showed that ThgI catalyses the condensation of acetyl-CoA and methylmalonyl-CoA to 2-methylacetoacetyl-CoA (MAA-CoA), ThgK catalyses NADPH-dependent reduction of MAA-CoA to 3-hydroxy-2-methylbutyryl-CoA (HMB-CoA) and ThgH catalyses the dehydration of HMB-CoA to angelyl-CoA (AN-CoA). This is the first report on the elucidation of the enzymatic formation of AN-CoA.

A new natural product, designated iminimycin A, was isolated from the cultured broth of a streptomycin-producing microbial strain, Streptomyces griseus OS-3601, via a physicochemical screening method using HP-20, silica gel and ODS column chromatographies and subsequent preparative HPLC. Iminimycin A is an indolizidine alkaloid, containing of an unusual iminium group and a cyclopropane ring with a triene side chain. The absolute configuration of iminimycin A was elucidated by NMR studies and electronic circular dichroism analysis. Iminimycin A shows anti-bacterial activity against Bacillus subtilis, Kocuria rhizophila and Xanthomonas campestris pv. orizae, and cytotoxic activity against HeLa S3 and Jurkat cells with IC50 values of 43 and 36 μM, respectively.

Iminimycin B, a novel indolizine alkaloid featuring a rare pyridinium, was isolated from the cultured broth of a streptomycin-producing strain, Streptomyces griseus OS-3601, through a physicochemical screening method. Its structure was elucidated on the basis of mass and NMR analyses. Stereochemical assignment of iminimycin B was archived by NMR studies, electronic circular dichroism (ECD) analysis, and advanced Marfey's method. (C) 2016 Elsevier Ltd. All rights reserved.

Our previous study revealed that aqueous extract of grape pomace obtained from a winemaking process could exert bactericidal action upon photo-irradiation via reactive oxygen species (ROS) formation. In the present study, we focused on chemical composition and prooxidative profile of the extract. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis showed that polyphenolic compounds including catechin monomers, dimers, trimers, and polyphenolic glucosides were contained. The polyphenol rich fraction used for the LC-ESI-MS analysis generated hydrogen peroxide (H2O2) upon photo-irradiation possibly initiated by photo-oxidation of phenolic hydroxyl group. That is, reduction of dissolved oxygen by proton-coupled electron transferred from the photo-oxidized phenolic hydroxyl group would form H2O2. The resultant H2O2 was then photolyzed to generate hydroxyl radical (•OH). The prooxidative profile of the extract in terms of •OH generation pattern upon photo-irradiation was similar to that of grape seed extract (GSE) as an authentic polyphenol product and (+)-catechin as a pure polyphenolic compound, and in all the three samples •OH generation could be retained during photo-irradiation for at least a couple of hours. The prooxidant activity of the photo-irradiated extract indicated by •OH yield was more potent than that of the photo-irradiated GSE and (+)-catechin, and this was well reflected in their bactericidal activity in which the photo-irradiated extract could kill the bacteria more efficiently than did the photo-irradiated GSE and (+)-catechin.

Two new compounds, nanaomycin F and G, were isolated by physicochemical screening method from cultured broth of Streptomyces rosa subsp. notoensis OS-3966, which is known to produce nanaomycin A, B, C, D, and E. Nanaomycin F is a new nanaomycin analog, a 4a-hydroxyl analog of nanaomycin B. Nanaomycin G has a unique skeleton with 1-indanone infused with a tetrahydropyran ring. Nanaomycin A possesses broad antimicrobial activity but nanaomycin F and G demonstrated no bioactivity against all bacteria and fungi tested in this study. In addition, in both nanaomycin F and G, the production of superoxide radicals was majorly decreased in comparison to nanaomycin A. It was considered that the antimicrobial properties were lost as a result of the decrease in production of the superoxide radicals.

Jietacin compounds are known to display potent nematocidal activity being 10-fold more active against the pine wood nematode (Bursaphelenchus lignicolus) than avermectin B1a. They consist of a unique functional vinylazoxy group. Herein, we disclose not only the isolation of novel analogs (jietacin C and D) but also a divergent and a 7-step total synthesis for jietacin A, B, C, and (S and R) D in 30-36% overall yield, incorporating reductive hydrazination, regioselective azoxy formation, and C C bond formation via acylation using Grignard reagents in the presence of vinylazoxy moiety at the final stage. In addition, we evaluated the nematocidal activity of jietacin A D and synthetic intermediates against Caenorhabditis elegans as a model nematode. (C) 2015 Elsevier Ltd. All rights reserved.

In the course of searching for insecticides from soil microorganisms, we found that a fermentation broth of the fungus, Trichoderma brevicompactum FKI-6324, produced Trichopolyn VI, a new peptaibol, which possessed significant insecticidal potential. Spectroscopic analysis showed the compound to be a new trichopolyn I derivative. This paper describes the isolation, structure elucidation and biological activity of trichopolyn VI.

Cytoprotective effects of short-term treatment with grape seed extract (GSE) upon human gingival fibroblasts (hGFs) were evaluated in relation to its antioxidant properties and compared with those of a water-soluble analog of vitamin E: trolox (Tx). GSE and Tx showed comparable antioxidant potential in vitro against di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH; a stable radical), hydroxyl radical (•OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Pretreatment or concomitant treatment with GSE for 1 min protected hGFs from oxidative stressors, including H2O2, acid-electrolyzed water (AEW), and 1O2, and attenuated the intracellular formation of reactive oxygen species induced by H2O2 and AEW. Tx also reduced the H2O2- and AEW-induced intracellular formation of reactive oxygen species, but showed no cytoprotective effects on hGFs exposed to H2O2, AEW, or 1O2. These results suggest that the cytoprotective effects of GSE are likely exerted independently of its antioxidant potential.

Under the hypothesis that photo-irradiated proanthocyanidin could accelerate wound healing through reactive oxygen species (ROS) formation, we examined the effect of proanthocyanidin on 3T3-L1 mouse fibroblasts with or without photo-irradiation. As a result, irrespective of presence or absence of photo-irradiation, only 1 min exposure of the cells to proanthocyanidin resulted in accelerated proliferation of the cells in a concentration-dependent manner. Similarly to proanthocyanidin, 1 min pretreatment with catechin, caffeic acid, and chlorogenic acid accelerated the proliferative response, but gallic acid, epicatechin gallate, epigallocatechin, and epigallocatechin gallate failed. If incorporated active ingredient such as proanthocyanidin for such a short time as 1 min accelerates the proliferation response, a bioassay was conducted by utilizing antioxidant potential of proanthocyanidin. That is, intracellular oxidation of 2',7'-dichlorodihydrofluorescin induced by H2O2 was significantly inhibited when the cells were pretreated with proanthocyanidin for 1 min, suggesting that incorporated proanthocyanidin into the cells exerted antioxidant effect. This was also supported by a liquid chromatography/mass spectrometry analysis in which incorporation of proanthocyanidin components such as catechin monomers and dimers into the cells within 1 min was confirmed. These results suggest that active polyphenolic compounds such as proanthocyanidin, catechin, caffeic acid, and chlorogenic acid incorporated into the cells in such a short time as 1 min could accelerate the proliferative response of the cells.

An actinomycete strain, K12-0602(T), was isolated from the root of a Helleborus orientalis plant in Japan. The 16S rRNA gene sequence of strain K12-0602(T) showed that it had a close relationship with members of the family Micromonosporaceae and the 16S rRNA gene sequence similarity values between strain K12-0602(T) and type strains of type species of 27 genera belonging to the family Micromonosporaceae were below 96.2%. MK-9 (H4) and MK-9 (H6) were detected as major menaquinones, and galactose, xylose, mannose and ribose were present in the whole-cell hydrolysate. The acyl type of the peptidoglycan was glycolyl. Major fatty acids were iso-C(15 : 0), iso-C(16 : 0), C(17 : 1)ω9c and anteiso-C(17 : 0). Phosphatidylethanolamine was detected as the phospholipid corresponding to phospholipid type II. The G+C content of the genomic DNA was 67 mol%. Analyses of the cell-wall peptidoglycan by TLC and LC/MS showed that it was composed of alanine, glycine, hydroxylglutamic acid and an unknown amino acid, which was subsequently determined to be 3,4-dihydroxydiaminopimelic acid using instrumental analyses, including NMR and mass spectrometry. On the basis of the phylogenetic analysis and chemotaxonomic characteristics, strain K12-0602(T) represents a novel species of a new genus in the family Micromonosporaceae, for which the name Rhizocola hellebori gen. nov., sp. nov. is proposed. The type strain of the type species is K12-0602(T) ( = NBRC 109834(T) = DSM 45988(T)). This is the first report, to our knowledge, of 3,4-dihydroxydiaminopimelic acid being found as a diamino acid in bacterial cell-wall peptidoglycan.

We have been continually searching for novel chemical compounds from culture broths of various actinomycetes using a physicochemical screening system. During the course of this program, we have previously reported the discovery of two new natural products, designated mangromicins A and B, discovered in a broth of a rare actinomycete strain, Lechevalieria aerocolonigenes K10-0216. Mangromicins have a unique and rare structure, a cyclopentadecane skeleton with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. New mangromicin analogs were isolated by using an improved production medium. As a consequence, six analogs, together with mangromicins A and B, were isolated from a cultured broth of L. aerocolonigenes K10-0216. We named them mangromicins D, E, F, G, H and I. All mangromicins showed radical scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and nitric oxide generated from LPS-stimulated RAW264.7 cells, a murine macrophage cell line. Among the analogs, mangromicins A and I showed the most potent DPPH radical scavenging activity and nitric oxide scavenging activity, respectively.

Two new cyclopentadecane antibiotics, named mangromicins A and B, were separated out from the culture broth of Lechevalieria aerocolonigenes K10-0216 by Diaion HP-20, silica gel and ODS column chromatography, and were finally purified by HPLC. The chemical structures of the two novel compounds were elucidated by instrumental analyses, including various NMR, MS and X-ray crystallography. Mangromicins A and B consist of cyclopentadecane skeletons with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. Mangromicins A and B showed in vitro antitrypanosomal activity with IC50 values of 2.4 and 43.4 μg ml(-1), respectively. The IC50 values of both compounds were lower than those of cytotoxicity against MRC-5 human fetal lung fibroblast cells.

Three new natural products, designated trehangelins A, B and C, were isolated by solvent extraction, silica gel and octadecylsilyl silica gel column chromatographies and subsequent preparative HPLC from the cultured broth of an endophytic actinomycete strain, Polymorphospora rubra K07-0510. The trehangelins consisted of a trehalose moiety and two angelic acid moieties. Trehangelins A (IC50 value, 0.1 mg ml(-1)) and C (IC50 value, 0.4 mg ml(-1)), with symmetric structures, showed potent inhibitory activity against hemolysis of red blood cells induced by light-activated pheophorbide a. However, trehangelin B, with an asymmetric structure, displayed only a slight inhibition (IC50 value, 1.0 mg ml(-1)).

We reported that generation of superoxide anion (O(2)(-)) was detected from conventional laboratory media. The generated O(2)(-) is non-enzymatic converted to hydroxyl radicals, which cause damage to lipids, proteins, and nucleic acids. However, the O(2)(-) generating mechanism from culture media is unclear. We considered that the O(2)(-) generation was implicated in a glycation reaction between reducing sugar and proteins, which is the early stage of Maillard reaction. It has been suggested that the glycated proteins, such as Schiff base and Amadori compounds, undergo a spontaneous autoxidation reaction, catalyzed by transition metal ions, involving the O(2)(-) generation. Therefore, we investigated the effect of Chelex 100 on the O(2)(-) generation from brain-heart-infusion (BHI) medium, which is a nutritional culture medium for bacteria. However, the O(2)(-) generation from the BHI medium treated with Chelex 100 was significantly increased in comparison to it treated without Chelex 100. The quantity of O(2)(-) generation from BHI medium was significantly increased by addition of glucose, and in alkaline environment as well as a glycation reaction model system that autoclaved a mixture solution of glucose and tryptophan. In addition, the O(2)(-) generation from BHI medium was significantly inhibited by pyridoxamine that is a Maillard reaction inhibitor. Therefore, it was suggested that the O(2)(-) generation from BHI medium is closely related to the glycation reaction of amide compounds such as proteins containing in the medium without the transition metals.

A time-course analysis of reactive oxygen species (ROS) generation in fertilized eggs of the devil stinger (Inimicus japonicus) from 0 h post-fertilization (hpf) to the early larval stage indicated that the ROS level was highest in the 22 hpf embryo, and declined thereafter. Phorbol myristate acetate (PMA) had no effect on ROS generation by the 22 hpf embryo, whereas PMA significantly increased larval ROS generation, suggesting that the ROS generation mechanisms of the 22 hpf embryo and larva are different at least in terms of PMA-responsiveness. Our results suggest the presence of a specific ROS generation system in devil stinger embryo which can be transitionally activated during embryogenesis.

We previously reported that superoxide anion was generated from glucose-peptone-meat extract medium. We found that superoxide anion was generated from other conventional laboratory media, Nutrient Broth, Tryptic Soy Broth, and Mueller Hinton Broth as well as glucose-peptone-meat extract medium. The glucose-peptone-meat extract medium consisting of 1% glucose, 0.5% peptone, 0.5% meat extract, and 0.3% NaCl showed the highest superoxide anion generation among the media tested. The meat extract and peptone, which are natural nutrient sources in the medium, showed an increase in absorbance intensity by WST-1 formazan products upon reaction with superoxide anion. The specific type of reactive oxygen species was measured by SOD-inhibitable reduction of cytochrome c and WST-1 (for superoxide anion), Fluoro H(2)O(2) kit (for hydrogen peroxide), hydroxyphenyl fluorescein (for hydroxyl radical), and Singlet Oxygen Sensor Green (for singlet oxygen). The generation of hydroxyl radical was also evaluated by ESR-spin trapping method. We detected superoxide anion, hydrogen peroxide, and hydroxyl radical but not singlet oxygen. These results suggest that the superoxide anion spontaneously generated from glucose-peptone-meat extract medium is finally converted to hydroxyl radical.

Based on a Blast search of 16S rRNA sequences of Streptomyces from marine environments of Nagasaki, Japan, 64 isolates showed the highest similarity scores with NBRC strains. Only 5 out of these 64 strains showed exactly the same biological profiles as the approximately 900 strains preserved at NBRC strains, while the remaining isolates showed different biological profiles. This suggests that the genus Streptomyces has the ability to produce a wide variety of unknown bioactive metabolites.

Sequence analysis of ketosynthase domain amplicons from Streptomyces bicolor NBRC 12746(T) revealed the presence of previously unreported type I polyketide synthases (PKS-I) genes. The clustering of these genes with the reference PKS-1 sequences suggested the possibility to produce a polyene compound similar to pimaricin. Thus, the cultured sample from NBRC 12746(T) was analyzed for the production of polyene compounds. The strain produced an antifungal compound which displayed the UV absorption spectrum of tetraene macrolides. The structure determination based on the spectroscopic analysis of the purified compound resulted in the identification of a novel pimaricin analog JBIR-13 (1). This study therefore strongly suggested that a careful analysis of PKS-I genes can provide valuable information in the search of novel bioactive compounds within a class predicted from phylogenetic analysis.

We examined the biological activities present in Streptomyces strains preserved at the National Institute of Technology and Evaluation Biological Resource Center, and found a metabolite of Streptomyces roseolilacinus NBRC 12815 that showed a potent anti-tyrosinase activity. The compounds with anti-tyrosinase activity were purified by several chromatographic procedures. Final HPLC analysis revealed at least two anti-tyrosinase compounds with different retention times (12815A and B). The identification of two anti-tyrosinase compounds was performed with instrumental analysis and database search. The results obtained suggest that the active compounds are SF 2583A and B. Compound 12815A (IC(50) values; about 9 microM) showed more potent tyrosinase inhibition than compound 12815B (IC(50) values; about 1086 microM). The only structural difference between 12815A and B is the presence of an additional chloric atom. In addition, the activity of 12815A was markedly decreased under acidic conditions, resulting in irreversible inactivation. However, the inactivated 12815A still exhibited residual activity when exposed to detergent, Tween 80. These results suggest that the chlorine and the hydration water are very important in the exertion of anti-tyrosinase activity by 12815A.

Penicillium strains (n=394) preserved at NBRC (the NITE Biological Resource Center) were compared as to groupings (11 species-clusters) based on phylogeny and the production of bioactive compounds. The strains in two clusters, of which P. chrysogenum and P. citrinum are representative, showed higher rates of positive strains with multi-biological activities.

The taxonomic distribution of Streptomyces species capable of producing bioactive compounds was investigated. Nine hundred and six strains were tested for the following four biological activities: antimicrobial, anti-tyrosinase, antioxidant, and hemolytic. Approximately 30% of strains tested showed antimicrobial activities, except for anti-Escherichia coli activity, which was present in only a few strains, while the rates of positivity for the anti-tyrosinase, antioxidant, and hemolytic activities were much lower. The distribution of Streptomyces strains capable of producing bioactive compounds was analyzed by the taxonomy based on 16S rRNA gene sequences. Moreover, the strains of Streptomyces hygroscopicus tested were divided into two clades in the phylogenetic tree, and all of the strains belonging to one clade showed antibacterial and antifungal activities. For detection of polyenes, the UV-visible spectra of metabolic extracts in the strains showing antifungal activities were measured. It was suggested that Streptomyces strains produce universal active compounds under different growth conditions. Further information on the relationship between the microbial taxonomy and the bioactive compounds produced would be useful for the utilization of industrial microorganisms.

Three hundred sixty-six Aspergillus strains preserved at the National Institute of Technology and Evaluation (NITE) were compared as to phylogenetic relationships (11 species-clusters) based on the DNA sequences of the D1/D2 domains of LSU rRNA and ITS regions, including the 5.8S rRNA and biological activities of their secondary metabolites. The results showed relatively well correlation between the phylogenetic distribution and the production of bioactive compounds, especially, antimicrobial activities.

About 800 strains of actinomycetes were isolated from marine environments around Nagasaki Prefecture, Japan. The isolates were compared with taxa and biological activities of their secondary metabolites. It is suggested that a variety of actinomycetes are isolated from different marine environments.

Heterocapsa circularisquama is known to cause lethal effect on bivalves, but toxic effect on fish has not been reported yet. Recently, we have found that H. circularisquama has potent light-dependent hemolytic toxins. Based on the chemical structural analysis, one of the hemolytic toxins named H2-a was found to be a novel porphyrin derivative with similar structure to pyropheophorbide a methyl ester (PME), a well-known photoactive hemolytic agent (Miyazaki et al., Aquatic Toxicol. 2005;73:382-393). To clarify the cytotoxic action mode of H2-a, we examined the effects of 1-12-a on HeLa cells in comparison with PME. The cytotoxicities of both reagents were strictly light dependent, and no significant cytotoxic effects including cellular morphological changes were induced without light illumination. The dose response curves revealed that H2-a showed stronger cytotoxicity to HeLa cells than PME. Fluorescence microscopic observation suggested that 1-12-a tends to accumulate in the plasma membrane, whereas PME seems to distribute entire cytoplasm. Although PME induced typical apoptotic nuclear morphological changes and DNA fragmentation in HeLa cells, no such apoptosis-inducing ability of H2-a was observed. Among the radical scavengers, histidine significantly inhibited the cytotoxic activity of H2-a, suggesting the involvement of singlet oxygen in the cytotoxicity. These results suggest that the cytotoxic mechanism of H2-a is necrotic rather than apoptosis differing from PME, even though these are structurally quite similar to each other. The relatively high affinity of 1-12-a to the plasma membrane might result in the potent and quick cytotoxicity without induction of apoptotic signal transduction. (C) 2008 Wiley Periodicals, Inc.

Prodigiosins are natural red pigments that have multi-biological activities. Recently, we discovered a marine bacterial strain, which produces a red pigment. Extensive two-dimensional nuclear magnetic resonance and mass spectrometry analysis showed that the pigment is a prodigiosin analogue (PG-L-1). Here, we investigated the effect of PG-L-1 on NADPH oxidase activity in macrophage cells. PG-L-1 significantly inhibited superoxide anion (O(2)(-)) production by phorbol 12-myristate 13-acetate (PMA)-stimulated RAW 264.7 cells, a mouse macrophage cell line. The ED(50) value was estimated to be approximately 0.3 microM. PG-L-1 had no direct scavenging effect on O(2)(-) generated by hypoxanthine/xanthine oxidase system in electron spin resonance-spin trapping determinations, suggesting that this compound directly acts on the O(2)(-) production system, NADPH oxidase, in macrophage cells. We further investigated the effect of PG-L-1 on the behaviour of the cytosolic components of the NADPH oxidase, p67(phox), p47(phox), p40(phox), Rac and protein kinase C (PKC), in PMA-stimulated RAW 264.7 cells. Although PG-L-1 showed no effect on the activation of PKC, the immunoblotting analysis using specific antibodies showed that PG-L-1 strongly inhibits the association of p47(phox) and Rac in the plasma membrane of PMA-stimulated RAW 264.7 cells. These results suggest that PG-L-1 inactivates NADPH oxidase through the inhibition of the binding of p47(phox) and Rac to the membrane components of NADPH oxidase.

A new indole-diterpene, JBIR-03 (1), was isolated from the fungus Dichotomomyces cejpii var. cejpii NBRC 103559 and its structure was determined based on the spectroscopic data. 1 exhibited anti-MRSA (methicillin-resistant Staphylococcus aureus) activity and antifungal activity against apple Valsa canker-causing fungus, Valsa ceratosperma, while it exhibited no toxicity towards human cancer cells.

A new member of the piericidin family, JBIR-02, was isolated from mycelium of Streptomyces sp. ML55 together with two known piericidin derivatives, piericidin A(1) and IT-143-B. The structure was determined on the basis of spectroscopic data. JBIR-02 inhibited nuclear export of beta-arrestin in HeLa cells at the concentration of 20 microM.

A new polypropionate alloaureothin (1) possessing a nitro group, together with a known polypropionate aureothin (2), was isolated from mycelium of Streptomyces sp. MM23. The structure was determined on the basis of spectroscopic data. 1 exhibited growth inhibitory effect against human fibrosarcoma HT1080 cells with an IC50 value of 30 microM.

Background: Reactive oxygen species (ROS) released from inflammatory cells constitute one of the critical causative factors in inflammatory skin diseases such as seborrhoeic dermatitis and atopic dermatitis. Objectives: To investigate inhibitory effects of ketoconazole (KCZ) and ciclopiroxolamine (CPO), both of which have been used for the treatment of seborrhoeic dermatitis, on ROS released from inflammatory cells. Methods: The methyl-Cypridina-luciferin analogue-dependent chemiluminescence method was employed for the detection of ROS production by phorbol 12-myristate 13-acetate (PMA)-stimulated inflammatory cells. Moreover, the radical scavenging activities of both agents were examined by using a hypoxanthine-xanthine oxidase system and the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). NADPH oxidase activity was determined in particulate (membrane) fractions prepared from PMA-stimulated RAW 264·7 cells, a macrophage-like cell line. Results Both of these antifungal agents inhibited PMA-stimulated ROS production. However, only CPO significantly scavenged both ROS generated by the hypoxanthine-xanthine oxidase system and DPPH, and the scavenging activity of CPO seemed to act on ROS other than superoxide anions. Although KCZ inhibited PMA-stimulated ROS production, it did not show radical-scavenging activities. The inhibition of ROS production by KCZ is probably attributable to the inhibition of NADPH oxidase activity. Conclusions: The mechanism of the inhibitory action of KCZ against PMA-stimulated ROS production is distinct from that of CPO. Knowledge of the inhibitory or scavenging effects of both antifungal agents on ROS released from inflammatory cells may be useful in developing a therapeutic strategy for dermatitis. © 2007 The Authors.

Raphidophycean flagellates, Chattonella marina and C. ovata, are harmful red tide phytoplankters
blooms of these phytoplankters often cause severe damage to fish farming. Previous studies have demonstrated that C. marina and C. ovata continuously produce reactive oxygen species (ROS) such as superoxide anion (O2-) hydrogen peroxide (H2O2) under normal growth conditions, and an ROS-mediated toxic mechanism against fish and other marine organisms has been proposed. Although the exact mechanism of ROS generation in these phytoplankters still remains to be clarified, our previous study suggested that NADPH oxidase-like enzyme located on the cell surface of C. marina may be involved in O2- generation. To investigate the localization of O2- and H 2O2 generation in C. marina and C. ovata, we employed 2-methyl-6(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one and 5-(and-6)-carboxy-2′,7′-dichlorodihydrodihydrofluorescein dictate, acetyl ester, which are specific fluorescent probe for detecting O 2- and H2O2, respectively. Observation by fluorescence microscopy of live phytoplankters incubated with each probe revealed that O2- is mainly generated on the cell surface, whereas H2O2 is generated in the intracellular compartment in these phytoplankters. When the cells were ruptured by ultrasonic treatment, O2- levels of C. marina and C. ovata decreased significantly, whereas a few times higher levels of H 2O2 were detected in the ruptured cell suspensions when compared with the levels of the live cell suspension. In immunoblotting analysis, the protein recognized by anti-human gp91 phox was detected in both species. These results suggest that, in both phytoplankters, the underlying mechanisms of O2- and H2O2 generation may be distinct and such systems are independently operating in the cells. © The Author 2007. Published by Oxford University Press. All rights reserved.

Egg lecithin greatly reduced the antibacterial activities of chlorhexidine gluconate (CHG). By using this antagonistic action, slower-growing bacteria, for which a longer incubation time was needed for its growth, were isolated from marine soils. The growth of relatively faster-growing bacteria, which formed visible colonies during a 3 day incubating period, was significantly inhibited by treatment with 8 mu g of CHG per mL for 10 min, whereas the addition of egg lecithin to a final concentration of 2%, after the treatment, had no significant effect on the viability of slower-growing bacteria.

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.

A marine gammaproteobacterium, strain MS-02-063, was able to kill Chattonella marina, a noxious red tide phytoplankton. However, the algicidal activity of bacterial cells washed with the planktonic medium was significantly reduced. These results suggest that strain MS-02-063 produces an extracellular substance, the pigment, PG-L-1, that showed a potent algicidal effect on C. marina. The LD50 value of PG-L-1 was calculated to be approximately 8.5 mu g ml(-1). At the approximate LD50 concentration of 10 mu g ml(-1), a morphological change, which seemed to be due to the inhibition of cell division, was observed in C. marina. Almost all cells of C. marina were destroyed readily at 100 mu g ml(-1) of PG-L-1, and the cytostatic activity of PG-L-1 against this phytoplankton was observed at a concentration of 1 mu g ml(-1) during the 5 d of incubation. A sublethal concentration of PG-L-1 of 10 mu g ml(-1) significantly inhibited the reactive oxygen species (ROS) production by C. marina. ROS production has been previously reported to be essential for normal growth of C. marina (Oda et al. 1995; Biosci Biotechnol Biochem 59:2044-2048). Therefore, the inhibitory effect of PG-L-1 on ROS production may lead to growth inhibition of C. marina, at least in part. The pigment, PG-L-1, may be a useful compound not only as an applicable agent for the mitigation of harmful algal blooms, but also as an experimental tool to analyse the ROS production system in a red tide phytoplankton such as C. marina.

Strain MS-02-063, gamma-proteobacterium, isolated from a coast area of Nagasaki, Japan, produced a red pigment which belongs to prodigiosin members. This pigment, PG-L-1, showed potent algicidal activity against various red tide phytoplanktons in a concentration-dependent manner. An understanding of a mechanism of PG-L-1 production by this marine bacterium may yield important new insights and strategies for preventing blooms of harmful flagellate algae in natural marine environments. Therefore, we analyzed the mechanisms of PG-L-1 production. In our previous study, the pigment production by this marine bacterium was completely inhibited at 1.56 microg/ml of erythromycin or 3.13 microg/ml of chloramphenicol, while minimal inhibitory concentrations for cell growth of erythromycin and chloramphenicol against this bacterium were >100 and 25 microg/ml, respectively. It is interesting to note that the ability of the pigment production in erythromycin-treated bacterium recovered by an addition of homoserine lactone. In fact, the pigment production was inhibited by beta-cyclodextrin that inhibits autoinducer activities by a complex with N-acyl homoserine lactones. N-acyl homoserine lactones with autoinducer activities are ubiquitous bacterial signaling molecules that regulate gene expression in a cell density dependent process known as quorum sensing. Therefore, it was suggested that PG-L-1 produced by strain MS-02-063 is controlled by the homoserine lactone quorum sensing. It is speculated that this quorum sensing is involved in the production of algicidal agents of other marine bacteria. This bacterium and other algicidal bacteria might be concerned in regulating the blooms of harmful flagellate algae through the quorum sensing system.

In aqua-cultural industry, the seed production of devil stinger, a valuable fish in Japan, has not succeeded yet due to the cryptogenic mass mortality. We found that survival rate of the larvae of devil stinger increased by the addition of green tea extract rich in catechin into rearing tank. Generation of reactive oxygen species (ROS) was detected in the embryo of devil stinger by chemiluminescence analysis under the normal growth conditions without addition of specific stimulants. Even in the unfertilized egg, certain level of ROS was detected. ROS were continuously detected during the development from fertilized egg to larva and tended to increase gradually. Observation of embryos and post-hatching larvae with hypersensitive photon-counting microscopy indicated that ROS were produced on the surface of embryo and the head region of larva especially peripheries of eyes. When the embryo proteins were analyzed by immunoblotting using antibody against the human neutrophil cytochrome b558 large subunit (gp91 phox), a main band of approximately 91 kDa was detected, suggesting the presence of NADPH oxidase-like ROS generating system in the embryo of devil stinger. After treatment with streptomycin and penicillin G for 1 day, the level of ROS production in larvae decreased with increase in the survival rate of larvae. Our results suggest that devil stinger has ROS generation system that is already activated at fairly early stage of development before the maturation of usual immune system.

Recently we discovered a bacterial strain (MS-02-063) that produces large amounts of red pigment (PG-L-1). Among the cell lines tested, U937 cells showed the highest susceptibility to PG-L-1 toxicity. PG-L-1 induced typical apoptotic nuclear morphological changes, and single cell gel electrophoresis revealed that PG-L-1 caused DNA fragmentation in U937 cells. In PG-L-1 treated U937 cells, the acidic compartment such as lysosomes disappeared, suggesting that PG-L-1-induced disorder of intracellular pH compartmentalization might trigger apoptotic signal. Since p38 MAP kinase inhibitor specifically prevented the PG-L-1 mediated cell death, p38 MAP kinase may be involved in the cytotoxic mechanism. In fact, immunoblot analysis of p38 MAP kinase revealed that phosphorylation of p38 MAP kinase occurred in PG-L-1-treated U937 cells. In addition to the activity to induce apoptotic cell death as reported in several PG family members, our chemiluminescence analysis suggested that PG-L-1 inhibited superoxide generation by 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated U937 cells in a dose-dependent manner. Since PG-L-1 had no effect on the chemiluminescence response caused by xanthine oxidase/hypoxanthine system, PG-L-1 acts on the enzyme system responsible for O(2)(-) generation rather than direct scavenging toward O(2)(-). Our results suggest that PG-L-1 causes multiple biochemical effects on the target cells such as increase in pH in acidic intracellular compartment, activation of p38 MAP kinase, inhibition of O(2)(-) generation, and eventually induces apoptotic cell death.

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.

We compared the abilities of alginate polymers having different molecular sizes and compositions to induce the secretion of tumor necrosis factor (TNF)-alpha in RAW264.7 cells. The molecular sizes and alpha-L-guluronate/beta-D-mannuronate (M/G) ratios of highly purified alginate polymers used in this study were 9000-38 000 and 1.50-3.17, respectively. Among the alginates tested, I-S, which had the highest molecular weight, showed the most potent TNF-alpha-inducing activity. The M/G ratio also seemed to influence this activity, and, among alginates with similar molecular sizes, alginates with a higher M/G ratio tended to show higher activity. Interestingly, the enzymatic depolymerization of I-S with bacterial alginate lyase resulted in a dramatic increase in the TNF-alpha-inducing activity. Such an effect of enzymatic digestion was also observed in a relatively low-molecular-weight alginate (ULV-3), which originally had very low TNF-alpha-inducing activity. Furthermore, the inhibition profiles of the TNF-alpha-inducing activity of enzymatically digested I-S shown by three specific mitogen-activated protein (MAP) kinase inhibitors differed from those of intact I-S. These results suggest that the underlying mechanism of the TNF-alpha-inducing activity of enzymatically depolymerized alginate oligomers is not necessarily the same as that of original alginate polymer.

We have previously found that the methanol extract prepared from Heterocapsa circularisquama, a harmful red rid dinoflagellate, showed light-dependent hemolytic activity toward rabbit erythrocytes. Interestingly, the cytotoxicity of the extract against HeLa cells was also strictly light-dependent, and no significant toxic effect was observed even at very high concentration in the dark. In this study, the hemolytic agents present in the extract were purified by several chromatographic procedures. Final HPLC analysis revealed that there are at least two hemolytic toxins with different retention times (H2-a and H3-a), which have similar absorption spectra. Although H3-a fraction was contaminated by a trace amount of H2-a, H2-a fraction gave a single peak on a three-dimensional chromatogram. The results of fast atom bombardment (FAB)-mass spectrometry (MS) and electrospray ionization quadrupole time-of-flight (ESI Q-TOF) mass spectrometry analyses suggested that the molecular weight of H2-a was 566. Since nuclear magnetic resonance analysis indicated the presence of a pyrrole ring in H2-a molecule, H2-a may be a porphyrin derivative with similar structure of pyropheophorbide a methyl ester, a well-known photosensitizing hemolytic compound. However, some structural and physicochemical differences were observed between H2-a and pyropheophorbide a methyl ester. Since H2-a showed potent photosensitizing cytotoxicity toward HeLa cells in a concentration-dependent manner as well as hemolytic activity, H2-a may be a novel porphyrin derivative with photosensitizing hemolytic cytotoxicity.

Chattonella marina (C. marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C. marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.

Prodigiosins (PGs) are known to be a family of natural red pigments, characterized by a common pyrrolydipyrrolylmethane skeleton structure with a C-4 methoxy group, and some of these pigments have been isolated from some microorganisms. Members of the PG family have been reported to show several biological activities, such as immunosuppressive and cytotoxic activities. Recently, we discovered a bacterial strain (MS-02-063), from our microbial library, that produces large amounts of a PG analogue (PG-L-1). In this study, we examined the anti-Trichophyton activity of PG-L-1 (produced by strain MS-02-063) against clinically isolated Trichophyton spp., by a method using stratum corneum epidermis (SCE) of the Yucatan micropig, which is suitable for estimating the antifungal activity of drugs in vitro. In the National Committee for Clinical Laboratory Standards (NCCLS) method, PG-L-1 showed potent antifungal activity against nine clinically isolated strains of Trichophyton spp., although the minimum inhibitory concentration (MIC) values were slightly higher than those of bifonazole. In spite of the lower efficiency of PG-L-1 transfer into SCE from medium than that of bifonazole, PG-L-1 transferred into SCE showed more potent antifungal activity than bifonazole, at lower concentrations.

Recently we discovered a bacterial strain (MS-02-063) that produces large amounts of red pigment from coastal area of Nagasaki Prefecture, Japan. Comparative 16S rDNA gene sequencing analysis revealed that strain MS-02-063 was phylogenetically closely related to gamma-proteobacterium Hahella sp. MBIC 3957 that produces prodigiosin. However, some physiological and biochemical differences between strain MS-02-063 and Hahella sp. MBIC 3957 were observed. The red pigment (RP-063) produced by this isolate was highly purified from the culture supernatant. It was speculated that RP-063 might be prodigiosin-like pigment in physical properties and biological activities such as antibacterial and cytotoxic activity. Antibacterial activity of RP-063 was examined by an agar dilution method. The results indicated that RP-063 showed antibacterial activity for specific for pathogenic gram-positive bacteria such as Staphylococcus aureus. The potency of antibacterial activity against S. aureus was nearly equal to those of tetracycline. Moreover, RP-063 showed inhibition of the superoxide generation by 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated mouse macrophage RAW 264.7 cell line. Prodigiosin members have a wide variety of biological properties, including anticancer and antimalarial, etc. Especially, potent immunosuppressive properties have been reported for prodigiosin members with the mechanism of action different from that of the other well known immunosuppressors in atopic dermatitis therapy such as cyclosporin A, FK506 and rapamycin. It is suggested that RP-063 may be able to arrest the inflammation caused by superantigens secreted from S. aureus, which colonized skin on atopic dermatitis as well as suppression of activated lymphocyte proliferation and superoxide generation from leucocytes.

We found that CEL-I was a potent cytotoxic lectin. MDCK, HeLa, and XC cells were highly sensitive to CEL-I cytotoxicity and killed in a dose-dependent manner, whereas CHO, L929, and RAW264.7 cells were relatively resistant to CEL-I, and no significant toxicity was observed up to 10 microg/ml. Among these cell lines, MDCK cells showed the highest susceptibility to CEL-I cytotoxicity. A binding study using FITC-labeled CEL-I (F-CEL-I) revealed that the amounts of bound F-CEL-I on the sensitive cell lines were evidently greater than those on the resistant cell lines, suggesting that the different susceptibility of the cell lines to CEL-I cytotoxicity is partly explained by different efficiencies of binding of CEL-I to these cell lines. Interestingly, the cytotoxicity of CEL-I toward MDCK cells was more potent than those of other lectins such as WGA, PHA-L, and Con A, even though these lectins were capable of binding to MDCK cells at comparable levels to CEL-I. Since the cytotoxicity of CEL-I was strongly inhibited by GalNAc, the binding to cell surface specific carbohydrates is essential for the CEL-I cytotoxicity. The trypan blue dye exclusion test indicated that CEL-I caused a disorder of plasma membrane integrity as a relatively early event. CEL-I failed to induce the release of carboxyfluorescein (CF) from CF-loaded MDCK cells as seen for pore-forming hemolytic isolectin CEL-III, suggesting that the primary cellular target of CEL-I may be the plasma membrane, but its action mechanism differs from that of CEL-III. Although CEL-I induced dramatic cellular morphological changes in MDCK cells, neither typical apoptotic nuclear morphological changes nor DNA fragmentation was observed in CEL-I-treated MDCK cells even after such cellular changes. Our results demonstrated that CEL-I showed a potent cytotoxic effect, especially on MDCK cells, by causing plasma membrane disorder without induction of apoptosis.

Therapy for onychomycosis is difficult because a complete cure requires long-term treatment. Although strong systemic antifungal drugs are potent enough to achieve a cure, they often have side effects. Therefore, one approach is to develop a new topical antifungal drug to act directly on the nail tissue. In this study, we developed a new in vitro model for assessing antifungal activity using the human nail. An O-ring was fixed with silicon bond to the dorsal surface of the nail. An antifungal agent applied to the surface will penetrate the nail tissue. The ventral side of the nail was placed on an agar plate inoculated with conidia of Trichophyton mentagrophytes. Nail specimens were infected with the fungi from the agar, and a clear fungal colony formed on the agar surrounding the nail on the fifth day of cultivation. After 14 days, the fungal colony in the control groups was shown to be expanding over the entire nail. The fungal colony in the group treated with sodium pyrithione had disappeared. Although the in vitro antifungal activity of sodium pyrithione has poor potency among the agents used, it showed remarkable antifungal activity, as assessed by image analysis. This model enables one to evaluate the activity of a topical antifungal agent that has penetrated human nail tissue, and it may facilitate research on a topical agent for onychomycosis.

Two surface-active compounds, egg lecithin and polysorbate 80, usually used as the deactivators of various preservatives were tested whether they also counteract either or all of the three major topical antifungal drugs, bifonazole (BFZ), lanoconazole (LCZ) and terbinafine (TBF). Both egg lecithin and polysorbate 80, when added to culture media up to final concentrations of 1.0 and 0.7%, respectively, antagonized the anti-dermatophytic activity of the three drugs in a concentration-dependent manner. A greater extent of antagonistic action was exerted when the two deactivators combined at their maximal levels tested were added; MIC's of BFZ were increased more than 30-fold and those of LCZ and TBF more than 200-fold compared with the values obtained in the absence of the deactivators. Using the agar medium supplemented with the combined deactivators, culture studies were carried out with skin tissues specimens taken from guinea pigs whose feet were infected with dermatophytes and subsequently treated with 1% topical preparations of the three antifungal drugs. The experimental data from this animal study demonstrated that the combined deactivators-supplemented medium yielded increased numbers of fungi compared with the basal medium. It looks, therefore, likely that the fungal recovery on the former medium more correctly reflects to actual fungal burden in the infected lesions than the latter. All these results suggest that the combined deactivators-supplemented medium is more useful for mycological evaluation of therapeutic efficacy of imidazole and allylamine drugs against dermatophytoses in both preclinical and clinical studies.

Antifungal susceptibility testing under conditions close to clinical status is expected to provide more helpful information than that obtained by a conventional microdilution method. For this purpose, we developed a novel method to evaluate anti-Trichophyton mentagrophytes activity of antifungal agents in vitro by using disks of micropig stratum corneum epidermis (SCE). Basal agar medium containing K2HPO4, MgSO4, CaCl2 and three kinds of antibiotics. Bifonazole (BFZ), lanoconazole (LCZ) or terbinafine (TBF) was added to the basal agar medium to give serially doubling dilutions ranging from 0.0006 to 10 microg/ml. Five-hundred-microl portions of the agar media thus prepared were solidified in wells of flat-bottomed plates. SCE disks (6 mm in diameter) were placed on surfaces of the agar medium and 10(4) conidia of T. mentagrophytes were inoculated on each SCE disk. There was very good correlation between the initial concentration of the antifungal agents added to the basal agar medium (microg/ml) and the concentration of the agents impregnated into the SCE disks (microg/g) (r2>0.99). The minimum inhibitory concentration (MIC) values of BFZ, LCZ and TBF were respectively 26-, 10- and 78-times higher than those measured by the standard microdilution method. From the correlation between the concentration of the agents in the basal medium and that in the SCE disks, the above MIC values corresponded to the concentrations in SCE disks (microg/g), 832.95 for BFZ, 1.42 for LCZ and 8.87 for TBF. This novel method of antidermatophytic susceptibility testing using SCE would be useful as an in vitro screening of proper antimycotics for topical treatment of dermatophytosis.