Aurantoside L, a New Tetramic Acid Glycoside with Anti-Leishmanial Activity Isolated from the Marine Sponge Siliquariaspongia japonica.

A new tetramic acid glycoside, aurantoside L (1), was isolated from the sponge Siliquariaspongia japonica collected at Tsushima Is., Nagasaki Prefecture, Japan. The structure of aurantoside L (1) composed of a tetramic acid bearing a chlorinated polyene system and a trisaccharide part was elucidated using spectral analysis. Aurantoside L (1) showed anti-parasitic activity against L. amazonensis with an IC50 value of 0.74 µM.

Muscle cell proliferation using water-soluble extract from nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 for sustainable cultured meat production.

Muscle cell cultivation, specifically the culture of artificial meat from livestock-derived cells in serum-free media is an emerging technology and has attracted much attention. However, till now, the high cost of production and environmental load have been significant deterrents. This study aims to provide an alternate growth-promoting substance that is free from animal derivatives and lowers nitrogen pollution. We have extracted water-soluble compounds from the filamentous nitrogen-fixing cyanobacteria Anabaena sp. PCC 7120 by the ultrasonication method. The heat-inactivated and molecular weight separation experiments were conducted to identify the bioactive compound present in the extract. Finally, the compounds soluble in water (CW) containing the water-soluble pigment protein, phycocyanin as a bioactive compound, was added as a growth supplement to cultivate muscle cells such as C2C12 muscle cells and quail muscle clone 7 (QM7) cells to analyze the effectiveness of the extract. The results indicated that CW had a positive role in muscle cell proliferation. A three-dimensional (3-D) cell-dense structure was fabricated by culturing QM7 cells using the extract. Furthermore, the nitrogen-fixing cyanobacterial extract has vast potential for cultured meat production without animal sera in the near future.

Two Onnamide Analogs from the Marine Sponge Theonella conica: Evaluation of Geometric Effects in the Polyene Systems on Biological Activity.

Two previously unreported onnamide analogs, 2Z- and 6Z-onnamides A (1 and 2), were isolated from the marine sponge Theonella conica collected at Amami-Oshima Is., Kagoshima Prefecture, Japan. Structures of compounds 1 and 2 were elucidated by spectral analysis. Structure-activity relationships (SARs) for effects on histone modifications and cytotoxicity against HeLa and P388 cells were characterized. The geometry in the polyene systems of onnamides affected the histone modification levels and cytotoxicity.

Histone modification dynamics as revealed by multicolor immunofluorescence-based single-cell analysis.

Post-translational modifications on histones can be stable epigenetic marks or transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here, we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. In extracts of marine organisms, we also screened chemical compounds that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

Commemorative Issue in Honor of Professor Paul J. Scheuer.

This Special Issue is dedicated to the memory of Professor Paul J [...].

Preparation and Application of a Chemical Probe for Identifying the Targets of the Marine Cyclic Peptide Kapakahine A.

Marine organisms are a rich source of bioactive secondary metabolites. Although many marine natural products with bioactivities have been isolated, successful elucidation of their mechanisms of action remains limited. In this study, we prepared a probe molecule based on the marine cyclic peptide kapakahine A (1) by introducing a linker with an azide terminal group, which enables the introduction of fluorescent groups for the effective monitoring of subcellular localization, or coupling to affinity beads for the pull-down of target proteins. The results of LC/MS/MS measurements, ProteinPilot analysis, and Western blotting suggest that kapakahine A interacts with the mitochondrial inner membrane proteins PHB1, PHB2, and ANT2, which is consistent with the results of the subcellular localization analysis using a fluorescent probe.

Small-Scale Preparation of Fluorescently Labeled Chemical Probes from Marine Cyclic Peptides, Kapakahines A and F.

A number of bioactive marine natural products have been isolated so far, but it is still difficult to disclose their modes of action. In this study, we prepared fluorescently labeled chemical probes from the cytotoxic marine cyclic peptides kapakahines A (1) and F (2) to visualize their localization as the first step of the study of their modes of action. We used fluorescent dyes 3a or 3a/b (a 1:1 mixture of 3a and 3b) whose terminal N-hydroxysuccinimide (NHS) group can react with the free amino groups of kapakahines. The fluorescently labeled kapakahine A (Kap A-5-FL, 5a) stained P388 murine leukemia cells and HeLa human cervical cancer cells, while cells treated with fluorescently labeled kapakahine F (Kap F-5-FL, 6a) only weakly stained them. Further analysis of the confocal images of the stained cells with higher magnification (×100) indicated the localization of Kap A-5-FL (5a) in the cells. In this paper, we report the small-scale preparation and a new delivery method of fluorescent probes, as well as the application of these procedures to cell staining.

A Soft Spot for Chemistry-Current Taxonomic and Evolutionary Implications of Sponge Secondary Metabolite Distribution.

Marine sponges are the most prolific marine sources for discovery of novel bioactive compounds. Sponge secondary metabolites are sought-after for their potential in pharmaceutical applications, and in the past, they were also used as taxonomic markers alongside the difficult and homoplasy-prone sponge morphology for species delineation (chemotaxonomy). The understanding of phylogenetic distribution and distinctiveness of metabolites to sponge lineages is pivotal to reveal pathways and evolution of compound production in sponges. This benefits the discovery rate and yield of bioprospecting for novel marine natural products by identifying lineages with high potential of being new sources of valuable sponge compounds. In this review, we summarize the current biochemical data on sponges and compare the metabolite distribution against a sponge phylogeny. We assess compound specificity to lineages, potential convergences, and suitability as diagnostic phylogenetic markers. Our study finds compound distribution corroborating current (molecular) phylogenetic hypotheses, which include yet unaccepted polyphyly of several demosponge orders and families. Likewise, several compounds and compound groups display a high degree of lineage specificity, which suggests homologous biosynthetic pathways among their taxa, which identifies yet unstudied species of this lineage as promising bioprospecting targets.

Kakeromamide A, a new cyclic pentapeptide inducing astrocyte differentiation isolated from the marine cyanobacterium Moorea bouillonii.

Kakeromamide A (1), a new cyclic pentapeptide encompassing a thiazole ring moiety and a ß-amino acid, was isolated from the marine cyanobacterium Moorea bouillonii. Its structure was elucidated by the spectral analysis and the modified Marfey's method. Compound 1 induced differentiation of neural stem cells into astrocytes at the concentration of 10 µM.

Sameuramide A, a new cyclic depsipeptide isolated from an ascidian of the family Didemnidae.

Sameuramide A (1), a new cyclic depsipeptide encompassing one each of alanine, N-methyl alanine, N-methyl dehydroalanine, N,O-dimethyl threonine, phenyllactic acid, three ß-hydroxy leucines, and two propionates, was isolated from a didemnid ascidian collected at the northern part of Japan. The planar structure was established based on the interpretation of MS and NMR data. The absolute configuration of the subunits was determined by the advanced Marfey's method and the chiral LC-MS analysis. Compound 1 exhibited the activity of maintaining colony formation of murine embryonic stem (mES) cells without leukemia inhibitory factor (LIF). Down regulation of the gene expression of Krüppel-like transcription factor 4 (Klf4) indicated that 1 itself was not able to maintain the undifferentiated state of the mES cells. However, the expression levels of the marker genes (Nestin, T, Sox17) for three germ layers were upregulated in embryoid bodies (EBs) after treatment of 1 together with LIF, suggesting that 1 plays a supportive role for LIF in maintaining the multipotency of mES cells.

Identification of zinc finger transcription factor EGR2 as a novel acetylated protein.

EGR2 is a zinc finger transcription factor that regulates myelination in the peripheral nervous system and T cell anergy. The transcriptional activity of EGR2 is known to be regulated by its co-activators and/or co-repressors. Although the activity of transcription factors is generally regulated not only by interactions with co-regulators but also posttranslational modifications including acetylation, little is known about posttranslational modifications of EGR2. Here we show that EGR2 is a novel acetylated protein. Through immunoblotting analyses using an antibody that specifically recognizes the acetylated form of EGR2, CBP and p300 were identified as acetyltransferases, while HDAC6, 10 and SIRT1 were identified as deacetylases of EGR2. Although the NuRD complex containing HDAC1 and HDAC2 is known to associate with EGR2, the present study suggests that acetylation of EGR2 is regulated independently of NuRD.

In†Vivo Gold Complex Catalysis within Live Mice.

Metal complex catalysis within biological systems is largely limited to cell and bacterial systems. In this work, a glycoalbumin-AuIII complex was designed and developed that enables organ-specific, localized propargyl ester amidation with nearby proteins within live mice. The targeted reactivity can be imaged through the use of Cy7.5- and TAMRA-linked propargyl ester based fluorescent probes. This targeting system could enable the exploitation of other metal catalysis strategies for biomedical and clinical applications.

One-Pot Evolution of Ageladine†A through a Bio-Inspired Cascade towards Selective Modulators of Neuronal Differentiation.

A bio-inspired cascade reaction has been developed for the construction of the marine natural product ageladine A and a de novo array of its N1-substituted derivatives. This cascade features a 2-aminoimidazole formation that is modeled after an arginine post-translational modification and an aza-electrocyclization. It can be effectively carried out in a one-pot procedure from simple anilines or guanidines, leading to structural analogues of ageladine A that had been otherwise synthetically inaccessible. We found that some compounds out of this structurally novel library show a significant activity in modulating the neural differentiation. Namely, these compounds selectively activate or inhibit the differentiation of neural stem cells to neurons, while being negligible in the differentiation to astrocytes. This study represents a successful case in which the native biofunction of a natural product could be altered by structural modifications.

Synthesis and antileishmanial activity of the core structure of cristaxenicin A.

The core structure of cristaxenicin A having trans-fused dihydropyran and nine membered ring has been synthesized and evaluated the antileishmanial activity. The dihydropyran ring was synthesized by [4+2] cycloaddition reaction between an unsaturated aldehyde and a ß-alkoxy-α,ß-unsaturated ketone. The nine membered ring possessing α,ß-unsaturated aldehyde was constructed by the intramolecular NHK reaction followed by the Mitsunobu rearrangement. The racemic core structure of cristaxenicin A was evaluated the anti-leishmanial activity with an IC50 value of 2.4µM.

Assay methods for small ubiquitin-like modifier (SUMO)-SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system.

SUMOylation is a posttranslational process that attaches a small ubiquitin-like modifier (SUMO) to its target proteins covalently. SUMOylation controls multiple cellular processes through the recognition of SUMO by a SUMO-interacting motif (SIM). In this study, we developed assay systems for detecting noncovalent interactions between SUMO and SIM in cells using split-luciferase complementation. We applied a version of this assay to the detection of in vitro SUMO-SIM interactions using a bacterial expression system. These novel assays enable screening of inhibitors of SUMO-dependent protein-protein interactions, either in vivo or in vitro, in a high-throughput manner.

Proliferation of mammalian cells with Chlorococcum littorale algal compounds without serum support.

In recent years, serum-free medium for mammalian cell cultivation has attracted a lot of attention, considering the high cost of production and environmental load involved in developing the conventional animal sera. The use of alternative growth-promoting products in mammalian cell cultivation such as extracts from microalgae has proven to be quite beneficial and environmental-friendly. This research aims to cultivate mammalian cells with growth-promoting factors derived from Chlorococcum littorale. We have established a simple extraction using the ultrasonication method and applied the extract in place of serum on mammalian C2C12 cell lines, 3T3 cell lines, and CHO cell lines to compare and analyze the effectiveness of the extract. Cell passage was conducted in a suspended culture condition with the addition of the extract. The results indicate that the extract from microalgae shows a high proliferation rate in all cell lines without fetal bovine serum. Moreover, it is eco-friendly and has huge potential to replace the traditional cell culture system. It could be applied in the fields of regenerative medicine, gene/cell therapies, as well as cultured meat production.

iGEM as a human iPS cell-based global epigenetic modulation detection assay provides throughput characterization of chemicals affecting DNA methylation.

Chemical-induced dysregulation of DNA methylation during the fetal period is known to contribute to developmental disorders or increase the risk of certain diseases later in life. In this study, we developed an iGEM (iPS cell-based global epigenetic modulation) detection assay using human induced pluripotent stem (hiPS) cells that express a fluorescently labeled methyl-CpG-binding domain (MBD), which enables a high-throughput screening of epigenetic teratogens/mutagens. 135 chemicals with known cardiotoxicity and carcinogenicity were categorized according to the MBD signal intensity, which reflects the degree of nuclear spatial distribution/concentration of DNA methylation. Further biological characterization through machine-learning analysis that integrated genome-wide DNA methylation, gene expression profiling, and knowledge-based pathway analysis revealed that chemicals with hyperactive MBD signals strongly associated their effects on DNA methylation and expression of genes involved in cell cycle and development. These results demonstrated that our MBD-based integrated analytical system is a powerful framework for detecting epigenetic compounds and providing mechanism insights of pharmaceutical development for sustainable human health.

Halenaquinone, a chemical compound that specifically inhibits the secondary DNA binding of RAD51.

Mutations and single-nucleotide polymorphisms affecting RAD51 gene function have been identified in several tumors, suggesting that the inappropriate expression of RAD51 activity may cause tumorigenesis. RAD51 is an essential enzyme for the homologous recombinational repair (HRR) of DNA double-strand breaks. In the HRR pathway, RAD51 catalyzes the homologous pairing between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is the central step of the HRR pathway. To identify a chemical compound that regulates the homologous-pairing activity of RAD51, in the present study, we screened crude extract fractions from marine sponges by the RAD51-mediated homologous-pairing assay. Halenaquinone was identified as an inhibitor of the RAD51 homologous-pairing activity. A surface plasmon resonance analysis indicated that halenaquinone directly bound to RAD51. Intriguingly, halenaquinone specifically inhibited dsDNA binding by RAD51 alone or the RAD51-ssDNA complex, but only weakly affected the RAD51-ssDNA binding. In vivo, halenaquinone significantly inhibited the retention of RAD51 at double-strand break sites. Therefore, halenaquinone is a novel type of RAD51 inhibitor that specifically inhibits the RAD51-dsDNA binding.

Cristaxenicin A, an antiprotozoal xenicane diterpenoid from the deep sea gorgonian Acanthoprimnoa cristata.

A new xenicane diterpenoid, cristaxenicin A (1), has been isolated from the deep sea gorgonian Acanthoprimnoa cristata. The structure of 1 was elucidated on the basis of spectral analysis including NMR and MS. The absolute configuration of 1 was determined on the basis of quantum chemical calculation of CD spectra. Cristaxenicin A (1) showed antiprotozoal activities against Leishmania amazonensis and Trypanosoma congolense with IC(50) values of 0.088 and 0.25 µM, respectively.

Structure-related cytotoxic activity of derivatives from kulokekahilide-2, a cyclodepsipeptide in Hawaiian marine mollusk.

Kulokekahilide-2, a 26-membered cyclodepsipeptide, was isolated from Hawaiian marine mollusk and possessed potent cytotoxicity in mammalian tumor cells. In the present study, we synthesized kulokekahilide-2 and its derivatives and examined the structure-activity relationships of these peptides in human cancer cells (A549, K562, and MCF7 cells). This study demonstrated that the cyclization of depsipeptide and the chirality of the 21 position in Ala in kulokekahilide-2 were important for its cytotoxic property and that addition of halogen at the para position of phenyl group in the 24-D-MePhe in kulokekahilide-2 as well as some derivatives remarkably increased their cytotoxicity in human cancer cells. These results suggest that the modifications of 24-D-MePhe in kulokekahilide-2, preserving its cyclization and the chirality at the 21-position, are promising strategy for exploring new derivative of kulokekahilide-2 as anti-tumor drug.