Mebamamide C, a deoxy analogue of mebamamides in Bryopsis marine green algae and Elysia sacoglossan mollusks.

Kahalalides, originally isolated from the sacoglossan mollusk Elysia rufescens, have been found in various Elysia and Bryopsis species, with over 20 variants identified to date. These compounds are biosynthesized by Candidatus Endobryopsis kahalalidefaciens within Bryopsis species. In this study, we report the isolation and structural determination of a new cyclic depsipeptide, mebamamide C (1), from Bryopsis sp. The planar structure was determined by spectroscopic data analyses, and the absolute configurations were determined using Marfey's method and modified Mosher's method. Additionally, our study explores the chemical relationship between Bryopsis algae and Elysia mollusks. The individual chemical profiles of these marine organisms highlight a fascinating aspect of marine chemical ecology. The distinct, species-specific chemical profiles observed in Elysia species imply the possibility of a symbiotic relationship with the kahalalide-producing bacteria.

Structure Determination of Kahalalide Analogues Based on Metagenomic Analysis of a Bryopsis sp. Marine Green Alga.

Two kahalalide analogues were isolated from a Bryopsis sp. marine green alga. Even though our initial structure determination of the peptides by NMR and MS identified them as kahalalide Z1 (KZ1; 3) and Z2 (KZ2; 4), the absolute configuration of the Thr residues by Marfey's analysis was different from those found in kahalalide F (KF), 3, and 4. To ascertain the absolute configuration of the amino acid residues genetically, we conducted a metagenomic analysis for symbiotic bacteria in the alga, leading to the biosynthetic gene cluster (BGC) responsible for producing the kahalalides named kahalalides Z3 (KZ3; 1) and Z4 (KZ4; 2). The identification of amino acid residues based on the A-domain suggested these peptides possess the amino acid sequence d-allo-Thr-l-Val-l-Val-d-Val residues at the N-terminus, instead of the d-Val-l-Thr-l-Val-d-Val residues found in KF, 3, and 4. The N-terminal amino acid sequence including absolute configuration was unambiguously determined by a comparison of LCMS data of synthetic tetrapeptides and the hydrolysates derived from 1 and 2. This structural difference is caused by swapping the substrate specificities of the first two A-domains.

Loss of DIAPH3, a Formin Family Protein, Leads to Cytokinetic Failure Only under High Temperature Conditions in Mouse FM3A Cells.

Cell division is essential for the maintenance of life and involves chromosome segregation and subsequent cytokinesis. The processes are tightly regulated at both the spatial and temporal level by various genes, and failures in this regulation are associated with oncogenesis. Here, we investigated the gene responsible for defects in cell division by using murine temperature-sensitive (ts) mutant strains, tsFT101 and tsFT50 cells. The ts mutants normally grow in a low temperature environment (32 °C) but fail to divide in a high temperature environment (39 °C). Exome sequencing and over-expression analyses identified Diaph3, a member of the formin family, as the cause of the temperature sensitivity observed in tsFT101 and tsFT50 cells. Interestingly, Diaph3 knockout cells showed abnormality in cytokinesis at 39 °C, and the phenotype was rescued by re-expression of Diaph3 WT, but not Diaph1 and Diaph2, other members of the formin family. Furthermore, Diaph3 knockout cells cultured at 39 °C showed a significant increase in the level of acetylated α-tubulin, an index of stabilized microtubules, and the level was reduced by Diaph3 expression. These results suggest that Diaph3 is required for cytokinesis only under high temperature conditions. Therefore, our study provides a new insight into the mechanisms by which regulatory factors of cell division function in a temperature-dependent manner.

Identification of GA-Binding Protein Transcription Factor Alpha Subunit (GABPA) as a Novel Bookmarking Factor.

Mitotic bookmarking constitutes a mechanism for transmitting transcriptional patterns through cell division. Bookmarking factors, comprising a subset of transcription factors (TFs), and multiple histone modifications retained in mitotic chromatin facilitate reactivation of transcription in the early G1 phase. However, the specific TFs that act as bookmarking factors remain largely unknown. Previously, we identified the "early G1 genes" and screened TFs that were predicted to bind to the upstream region of these genes, then identified GA-binding protein transcription factor alpha subunit (GABPA) and Sp1 transcription factor (SP1) as candidate bookmarking factors. Here we show that GABPA and multiple histone acetylation marks such as H3K9/14AC, H3K27AC, and H4K5AC are maintained at specific genomic sites in mitosis. During the M/G1 transition, the levels of these histone acetylations at the upstream regions of genes bound by GABPA in mitosis are decreased. Upon depletion of GABPA, levels of histone acetylation, especially H4K5AC, at several gene regions are increased, along with transcriptional induction at 1 h after release. Therefore, we proposed that GABPA cooperates with the states of histone acetylation to act as a novel bookmarking factor which, may negatively regulate transcription during the early G1 phase.

Neighbors' death is required for surviving human adenocarcinoma PC-9 cells in an early stage of gefitinib treatment.

Acquired drug resistance is a major problem in chemotherapy, and understanding of the mechanism, by which naïve cells defend themselves from drugs when the cells exposed to the drugs for the first time, may provide a solution of the problem. Gefitinib is an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, and used as an anticancer drug; however, gefitinib treatment may sometimes lead cancer cells gradually into a gefitinib-tolerance. Here we describe that human adenocarcinoma PC-9 cells even under the presence of gefitinib were able to survive by activating another signaling pathway involving fibroblast growth factor receptor (FGFR) and its signaling molecule, FGF2; and further suggest that the FGF2 for initiating the pathway might be supplied from neighboring cells which were killed by gefitinib, i.e., the survival might be founded on neighbors' sacrifice in an early stage of gefitinib treatment. Our findings suggested that whether cells had a chance to encounter to survival factors such as FGF2 soon after gefitinib treatment might be an important crossroads for the cells for survival and for gaining a gefitinib tolerance.

Identification of preferentially reactivated genes during early G1 phase using nascent mRNA as an index of transcriptional activity.

During mammalian mitosis, transcription is silenced due to dissociation of transcription factors from DNA and chromosome condensation. At the end of mitosis, transcription is reactivated through chromosome relaxation and reloading of these factors to the DNA. Early G1 genes, which are preferentially reactivated during M/G1 transition, are important for maintenance of cellular function and are known to be strictly regulated. As only few early G1 genes have been identified to date, screening for early G1 genes by genome-wide analysis using nascent mRNA could contribute to the elucidation of the regulatory mechanisms during early G1. Here, we performed a detailed expression analysis for the M/G1 transition of mammalian cells by microarray analysis of nascent mRNA, and identified 298 early G1 genes. Analysis of these genes provides two important insights. Firstly, certain motifs are enriched in the upstream sequences of early G1 genes; from this we could predict candidate cognate transcription factors, including Sp1, which is recruited to the DNA in the early G1 phase. Secondly, we discovered that neighboring genes of early G1 genes were also frequently up-regulated in the G1 phase. Information about these numerous newly identified early G1 genes will likely contribute to an understanding of the regulatory mechanisms of the early G1 genes.

MiR-199-3p enhances muscle regeneration and ameliorates aged muscle and muscular dystrophy.

Parabiosis experiments suggest that molecular factors related to rejuvenation and aging circulate in the blood. Here, we show that miR-199-3p, which circulates in the blood as a cell-free miRNA, is significantly decreased in the blood of aged mice compared to young mice; and miR-199-3p has the ability to enhance myogenic differentiation and muscle regeneration. Administration of miR-199 mimics, which supply miR-199-3p, to aged mice resulted in muscle fiber hypertrophy and delayed loss of muscle strength. Systemic administration of miR-199 mimics to mdx mice, a well-known animal model of Duchenne muscular dystrophy (DMD), markedly improved the muscle strength of mice. Taken together, cell-free miR-199-3p in the blood may have an anti-aging effect such as a hypertrophic effect in aged muscle fibers and could have potential as a novel RNA therapeutic for DMD as well as age-related diseases. The findings provide us with new insights into blood-circulating miRNAs as age-related molecules.

Maternal protein restriction induces renal AT2R promoter hypomethylation in salt-sensitive, hypertensive rats.

SCOPE: We previously demonstrated that protein restriction in utero induced salt-sensitive hypertension and changed renal levels of angiotensin type 2 receptor (AT2R) in Stroke-Prone Spontaneously Hypertensive Rat (SHRSP). Here, we investigated if this characteristic alteration of AT2R is related to AT2R DNA methylation profiles. METHODS AND RESULTS: First, we examined the relation between AT2R DNA methylation and its promoter activity in vitro. Luciferase assays revealed a negative correlation between these two variables. Next, we fed SHRSP dams and grand-dams a control 20% casein diet or a 9% casein diet during pregnancy. Adult offspring and grand-offspring were supplied either water or 1% saline solution for 2 weeks. Renal AT2R promoter DNA near the TATA-box was hypomethylated, mRNA expression was suppressed, and protein expression tended to be higher, in adult offspring of mothers fed a low casein diet. Moreover, adult grand-offspring exhibited high blood pressure after salt loading, along with suppressed transcription of AT2R mRNA and elevated translated protein. CONCLUSIONS: Under a fetal environment of protein restriction, the increase in protein expression due to hypomethylation of the AT2R promoter region occurs as a response to increased salt sensitivity, and controlling this mechanism may be important for the prevention of hypertension.

Three multi-allelic gene pairs are responsible for self-sterility in the ascidian Ciona intestinalis.

Many hermaphroditic organisms possess a self-incompatibility system to avoid inbreeding. Although the mechanisms of self-incompatibility in flowering plants are well known, little is known about the mechanisms of self-sterility in hermaphroditic marine invertebrates. Ascidians are hermaphroditic sessile marine invertebrates that release sperm and eggs into the surrounding seawater. Several species, including Ciona intestinalis type A (Ciona robusta), exhibit strict self-sterility. In a previous study, we found that the candidate genes responsible for self-sterility in Ciona reside in chromosome 2q (locus A) and chromosome 7q (locus B). Two pairs of multi-allelic genes, named s(sperm)-Themis-A and v(vitelline-coat)-Themis-A in locus A and s-Themis-B and v-Themis-B in locus B, are responsible for self-sterility. In this study, we identified a third multi-allelic gene pair, s-Themis-B2 and v-Themis-B2, within locus B that is also involved in this system. Genetic analysis revealed that the haplotypes of s/v-Themis-A, s/v-Themis-B and s/v-Themis-B2 play essential roles in self-sterility. When three haplotypes were matched between s-Themis and v-Themis, fertilization never occurred even in nonself crossing. Interestingly, gene targeting of either s/v-Themis-B/B2 or s/v-Themis-A by genome editing enabled self-fertilization. These results indicate that s/v-Themis-A, -B and -B2 are S-determinant genes responsible for self-sterility in the ascidian C. intestinalis type A.

Comprehensive Measurement of Gene Silencing Involving Endogenous MicroRNAs in Mammalian Cells.

MicroRNAs (miRNAs) are functional small noncoding RNAs that work as mediators in gene silencing and that play important roles in gene regulation. A number of miRNAs have been found and their expression profiles have been examined by means of various microarray systems and real-time polymerase chain reaction (PCR) systems. Conventional microarrays as well as real-time PCR are able to detect existing miRNAs, in which inactive miRNAs that hardly contribute to gene silencing may be also contained. Here, we describe a comprehensive miRNA bioassay system with reporter genes for the detection of active miRNAs that are present in the RNA-induced silencing complexes, and actually working as mediators in gene silencing.

NF-κB activation is an early event of changes in gene regulation for acquiring drug resistance in human adenocarcinoma PC-9 cells.

Gefitinib and erlotinib are epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). Although EGFR-TKIs are effective as anti-cancer drugs, cancer cells sometimes gain tolerance to the drugs. Previous studies suggested that the fibroblast growth factor receptor (FGFR)-signaling pathway could serve as compensation for the EGFR-signaling pathway inhibited by EGFR-TKIs. Our study further suggested that FGF2, a FGFR ligand, leaked out from naïve cells killed by gefitinib could initiate the FGFR-signaling pathway in surviving cells; i.e., altruistic survival may occur in naïve cells immediately after EGFR-TKI treatment. Altruistic survival may be temporal, and cells need to change their gene regulation toward gaining resistance to EGFR-TKIs. Changes in such gene regulation after EGFR-TKI treatment are poorly understood. In this study, we examined early events of such gene regulation changes in human adenocarcinoma PC-9 cells that are capable of changing their nature from susceptibility to resistance to EFGR-TKIs. Our study indicated that activation of nuclear factor-kappa B (NF-κB) occurred in the cells immediately after EGFR-TKI treatment and also by gene silencing against oncogenic EGFR; and, MG132 treatment for inhibiting NF-κB activation affected cell viability. Taken together, our findings (including the previous study) suggest that altruistic survival and NF-κB activation might be vital for initiating the acquisition of EGFR-TKI resistance.

Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis.

Multiple sclerosis (MS) is a T cell-mediated autoimmune disease of the central nervous system. Foxp3+ regulatory T (Treg) cells are reduced in frequency and dysfunctional in patients with MS, but the underlying mechanisms of this deficiency are unclear. Here, we show that induction of human IFN-γ-IL-17A-Foxp3+CD4+ T cells is inhibited in the presence of circulating exosomes from patients with MS. The exosomal miRNA profile of patients with MS differs from that of healthy controls, and let-7i, which is markedly increased in patients with MS, suppresses induction of Treg cells by targeting insulin like growth factor 1 receptor (IGF1R) and transforming growth factor beta receptor 1 (TGFBR1). Consistently, the expression of IGF1R and TGFBR1 on circulating naive CD4+ T cells is reduced in patients with MS. Thus, our study shows that exosomal let-7i regulates MS pathogenesis by blocking the IGF1R/TGFBR1 pathway.

Supplemental Treatment for Huntington's Disease with miR-132 that Is Deficient in Huntington's Disease Brain.

Huntington's disease (HD) is an intractable neurodegenerative disorder caused by mutant Huntingtin (HTT) proteins that adversely affect various biomolecules and genes. MicroRNAs (miRNAs), which are functional small non-coding RNAs, are also affected by mutant HTT proteins. Here, we show amelioration in motor function and lifespan of HD-model mice, R6/2 mice, by supplying miR-132 to HD brains using a recombinant adeno-associated virus (rAAV) miRNA expression system. miR-132 is an miRNA related to neuronal maturation and function, but the level of miR-132 in the brain of R6/2 mice was significantly lower than that of wild-type mice. Our miR-132 supplemental treatment, i.e., supplying miR-132 to the brain, produced symptomatic improvement or retarded disease progression in R6/2 mice; interestingly, it had little effect on disease-causing mutant HTT mRNA expression and its products. Therefore, the findings suggest that there may be a therapeutic way to treat HD without inhibiting and/or repairing disease-causing HTT genes and gene products. Although miR-132 supplement may not be a definitive treatment for HD, it may become a therapeutic method for relieving HD symptoms and delaying HD progression.

Glucocorticoid affects dendritic transport of BDNF-containing vesicles.

Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival, differentiation, and functions in the central nervous system (CNS). Because BDNF protein is sorted into secretory vesicles at the trans-Golgi network in the cell body after translation, transport of BDNF-containing vesicles to the secretion sites is an important process for its function. Here we examined the effect of dexamethasone (DEX), a synthetic glucocorticoid, on BDNF-containing vesicle transport and found that DEX decreased the proportion of stationary vesicles and increased velocity of the microtubule-based vesicle transport in dendrites of cortical neurons. Furthermore, DEX increased huntingtin (Htt) protein levels via glucocorticoid receptor (GR) activation, and reduction in the amount of Htt by a specific shRNA reversed the action of DEX on BDNF vesicle transport. Given that Htt protein is a positive regulator for the microtubule-dependent vesicular transport in neurons, our data suggest that glucocorticoid stimulates BDNF vesicle transport through upregulation of Htt protein levels.

Normalization of Overexpressed α-Synuclein Causing Parkinson's Disease By a Moderate Gene Silencing With RNA Interference.

The α-synuclein (SNCA) gene is a responsible gene for Parkinson's disease (PD); and not only nucleotide variations but also overexpression of SNCA appears to be involved in the pathogenesis of PD. A specific inhibition against mutant SNCA genes carrying nucleotide variations may be feasible by a specific silencing such as an allele-specific RNA interference (RNAi); however, there is no method for restoring the SNCA overexpression to a normal level. Here, we show that an atypical RNAi using small interfering RNAs (siRNAs) that confer a moderate level of gene silencing is capable of controlling overexpressed SNCA genes to return to a normal level; named "expression-control RNAi" (ExCont-RNAi). ExCont-RNAi exhibited little or no significant off-target effects in its treated PD patient's fibroblasts that carry SNCA triplication. To further assess the therapeutic effect of ExCont-RNAi, PD-model flies that carried the human SNCA gene underwent an ExCont-RNAi treatment. The treated PD-flies demonstrated a significant improvement in their motor function. Our current findings suggested that ExCont-RNAi might be capable of becoming a novel therapeutic procedure for PD with the SNCA overexpression, and that siRNAs conferring a moderate level of gene silencing to target genes, which have been abandoned as useless siRNAs so far, might be available for controlling abnormally expressed disease-causing genes without producing adverse effects.

Gene silencing mediated by endogenous microRNAs under heat stress conditions in mammalian cells.

Heat shock, sudden change in temperature, triggers various responses in cells for protecting the cells from such a severe circumstance. Here we investigated gene silencing mediated by endogenous microRNAs (miRNAs) in mammalian cells exposed to a mild hyperthermia, by means of miRNA activity assay using a luciferase reporter gene as well as miRNA expression analysis using a DNA microarray. Our findings indicated that the gene silencing activities involving miRNAs were enhanced without increasing in their expression levels under heat-stress conditions. Additionally, the gene silencing activity appeared to be independent of the cytoprotective action involving heat shock proteins that are immediately activated in heat-shocked cells and that function as molecular chaperons for restoring heat-denatured proteins to normal proteins. Our current findings suggested the possibility that gene silencing involving endogenous miRNAs might play a subsidiary role in heat-shocked cells for an aggressive inhibition of the expression of heat-denatured proteins.

Novel DNA microarray system for analysis of nascent mRNAs.

Transcriptional activation and repression are a key step in the regulation of all cellular activities. The development of comprehensive analysis methods such as DNA microarray has advanced our understanding of the correlation between the regulation of transcription and that of cellular mechanisms. However, DNA microarray analysis based on steady-state mRNA (total mRNA) does not always correspond to transcriptional activation or repression. To comprehend these transcriptional regulations, the detection of nascent RNAs is more informative. Although the nuclear run-on assay can detect nascent RNAs, it has not been fully applied to DNA microarray analysis. In this study, we have developed a highly efficient method for isolating bromouridine-labeled nascent RNAs that can be successfully applied to DNA microarray analysis. This method can linearly amplify small amounts of mRNAs with little bias. Furthermore, we have applied this method to DNA microarray analysis from mouse G2-arrested cells and have identified several genes that exhibit novel expression profiles. This method will provide important information in the field of transcriptome analysis of various cellular processes.