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The copy number of mitochondrial DNA (mtDNA) is decreased in most cancer types, including hepatocellular carcinoma (HCC), compared to normal counterparts. However, a decrease in mtDNA usually leads to defects in cell proliferation, which contradicts the robustness of cancer cell proliferation. In this study, we found that four out of seven HCC cell lines were of the mtDNA-less type. Interestingly, FOXM1, a member of the FOX transcription factor family, was highly expressed in a subset of them with proliferative potential maintained. B-MYB, a partner of FOXM1, was also expressed in the same cell lines. RNAi-mediated experiments demonstrated that when FOXM1/B-MYB was silenced in the cell lines, cell cycle-related genes were downregulated, while p21Cip1 was induced with senescence-associated β-galactosidase, resulting in G1/S cell cycle arrest. These results suggest that high expression of FOXM1/B-MYB is critical for sustaining cell proliferation in mtDNA-less cells. In addition, we found that high expression of FOXM1 was mediated by the deubiquitinating enzyme, OTUB1, in one cell line. Thus, interference with FOXM1/B-MYB expression, such as through OTUB1 inhibition, may induce a dormant state of senescence-like proliferation arrest in mtDNA-less cancer cells. This finding may be utilized for the development of precision medicine for relevant cancers.

Non-ribosomal peptide (NRP) natural products are one of the most promising resources for drug discovery and development because of their wide-ranging of therapeutic potential, and their behavior as virulence factors and signaling molecules. The NRPs are biosynthesized independently of the ribosome by enzyme assembly lines known as the non-ribosomal peptide synthetase (NRPS) machinery. Genetic, biochemical, and bioinformatics analyses have provided a detailed understanding of the mechanism of NRPS catalysis. However, proteomic techniques for natural product biosynthesis remain a developing field. New strategies are needed to investigate the proteomes of diverse producer organisms and directly analyze the endogenous NRPS machinery. Advanced platforms should verify protein expression, protein folding, and activities and also enable the profiling of the NRPS machinery in biological samples from wild-type, heterologous, and engineered bacterial systems. Here, we focus on activity-based protein profiling strategies that have been recently developed for studies aimed at visualizing and monitoring the NRPS machinery and also for rapid labeling, identification, and biochemical analysis of NRPS enzyme family members as required for proteomic chemistry in natural product sciences.

A hitherto unreported member of γ-alkylidenebutenolides in Melodorum fruticosum (Annonaceae), (4E)-6-benzoyloxy-7-hydroxy-2,4-heptadiene-4-olide, named as isofruticosinol (4) was isolated from the methanol extract of flowers, along with the known related butenolides, namely, the (4Z)-isomer (3) of 4, melodrinol (1), and its (4E)-isomer (2). To unambiguously determine the absolute configuration at the C-6 position in these butenolides, the first total syntheses of both enantiomers of 2–4 were achieved over 6–7 steps from commercially available D- or L-ribose (D- and L-5). Using the same protocol, both enantiomers of 1 were also synthesized. Based on chiral HPLC analysis of all synthetic compounds (S- and R-1–4), all naturally occurring butenolides were assigned as partial racemic mixtures with respect to the chiral center at C-6 (enantiomeric ratio, 6S/6R = ∼83/17). Furthermore, the melanogenesis inhibitory activities of S- and R-1–4 were evaluated, with all shown to be potent inhibitors with IC50 values i...

We present a simple and microscopic physical model that breaks the ergodicity. Our model consists of coupled classical harmonic oscillators, and the motion of the tagged particle obeys the generalized Langevin equation satisfying the second fluctuation dissipation theorem. It is found that although the nonergodicity strength, which is expected to detect the ergodicity breaking, for this model vanishes, the velocity auto correlation function of the tagged particle asymptotically oscillates. We analyze the model by using the molecular dynamics and the exact diagonalization as well as the rigorous mapping to the generalized Langevin equation. Our analysis reveals that the asymptotic oscillation is caused by a localized mode with an isolated frequency from the continuous phonon spectrum.

Matrix metalloproteinases (MMPs) are tissue‐remodeling enzymes involved in the processing of various biological molecules. MMPs also play important roles in cancer metastasis, contributing to angiogenesis, intravasation of tumor cells, and cell migration and invasion. Accordingly, unraveling the signaling pathways controlling MMP activities could shed additional light on cancer biology. Here, we report a molecular axis, comprising the molecular adaptor hydrogen peroxide‐inducible clone‐5 (HIC‐5), NADPH oxidase 4 (NOX4), and mitochondria‐associated reactive oxygen species (mtROS), that regulates MMP9 expression and may be a target to suppress cancer metastasis. We found that this axis primarily downregulates mtROS levels which stabilize MMP9 mRNA. Specifically, HIC‐5 suppressed the expression of NOX4, the source of the mtROS, thereby decreasing mtROS levels and, consequently, destabilizing MMP9 mRNA. Interestingly, among six cancer cell lines, only EJ‐1 and MDA‐MB‐231 cells exhibited upregulation of NOX4 and MMP9 expression after shRNA‐mediated HIC‐5 knockdown. In these two cell lines, activating RAS mutations commonly occur, suggesting that the HIC‐5–mediated suppression of NOX4 depends on RAS signaling, a hypothesis that was supported experimentally by the introduction of activated RAS into mammary epithelial cells. Notably, HIC‐5 knockdown promoted lung metastasis of MDA‐MB‐231 cancer cells in mice. The tumor growth of HIC‐5–silenced MDA‐MB‐231 cells at the primary sites was comparable to that of control cells. Consistently, the invasive properties of the cells, but not their proliferation, were enhanced by the HIC‐5 knockdown in vitro. We conclude that NOX4‐mediated mtROS signaling increases MMP9 mRNA stability and affects cancer invasiveness but not tumor growth., Matrix metalloproteinases (MMPs) are a group of tissue‐remodeling enzymes that have been linked to a variety of pathophysiological processes, including cancer metastasis. Here, Motoko Shibanuma and co‐authors provide new insights into the regulation of MMP9. They show that the molecular adaptor protein HIC‐5 suppresses the activation of NADPH oxidase 4 (NOX4), leading to downregulation of mitochondrial ROS levels, which in turn destabilizes MMP9 mRNA. They further demonstrate that this regulatory axis operates specifically in cancer cells harboring oncogenic mutations in H‐ or K‐ras, potentially unveiling a new therapeutic avenue for cancer therapy based on inhibition of MMPs.

Non-ribosomal peptide (NRP) natural products are one of the most promising resources for drug discovery and development because of their wide-ranging of therapeutic potential, and their behavior as virulence factors and signaling molecules. The NRPs are biosynthesized independently of the ribosome by enzyme assembly lines known as the non-ribosomal peptide synthetase (NRPS) machinery. Genetic, biochemical, and bioinformatics analyses have provided a detailed understanding of the mechanism of NRPS catalysis. However, proteomic techniques for natural product biosynthesis remain a developing field. New strategies are needed to investigate the proteomes of diverse producer organisms and directly analyze the endogenous NRPS machinery. Advanced platforms should verify protein expression, protein folding, and activities and also enable the profiling of the NRPS machinery in biological samples from wild-type, heterologous, and engineered bacterial systems. Here, we focus on activity-based protein profiling strategies that have been recently developed for studies aimed at visualizing and monitoring the NRPS machinery and also for rapid labeling, identification, and biochemical analysis of NRPS enzyme family members as required for proteomic chemistry in natural product sciences.

Adenylation (A) domains found in all nonribosomal peptide synthetase (NRPS) modules are essential catalytic components and function as gatekeepers to select amino acid building blocks during nonribosomal peptide biosynthesis. Leveraging the strict substrate recognition characteristics of these enzymes, we targeted the development of active site-directed proteomic probes for A domains in NRPSs that enable detection, isolation, identification, and enzymatic characterization of A domains in native proteomes. Here, we describe a general strategy for selective chemical labeling of individual A domains in NRPS enzymes using active site-directed proteomic probes coupled to 5′-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality. These probes selectively target individual A domains in natural product producer proteomes by ligand-directed protein labeling. The data demonstrate that these proteomic tools can greatly facilitate the molecular identification, functional characterization, and profiling of virtually any kind of A domains of NRPS enzymes in complex biological systems.

Phosphopantetheinylation is an essential post-translational protein modification to primary and secondary metabolic pathways that ensures bacterial cell viability and virulence, and it is used in the production of many pharmaceuticals. Traditional methods have not provided a comprehensive understanding of these modifications. By using chemical proteomic probes for adenylation and thiolation domains in nonribosomal peptide synthetases (NRPSs), chemoproteomics has been applied to survey and validate the cellular activity of 4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), which is a potent and selective small-molecule 4'-phosphopantetheinyl transferase (PPTase) inhibitor that attenuates secondary metabolism and viability of bacterial cells. ML267 inhibited Sfp-type PPTase and antagonized phosphopantetheinylation in cells, which resulted in a decrease in phosphopantetheinylated NRPSs and the attenuation of Sfp-PPTase-dependent metabolite production. These results indicate that this chemoproteomics platform should enable a precise interpretation of the cellular activities of Sfp-type PPTase inhibitors.

We performed X-ray diffraction and electrical resistivity measurement up to pressures of 5 GPa and the first-principles calculations utilizing experimental structural parameters to investigate the pressure-induced topological phase transition in BiTeBr having a noncentrosymmetric layered structure (space group P3m1). The P3m1 structure remains stable up to pressures of 5 GPa; the ratio of lattice constants, c/a, has a minimum at pressures of 2.5 - 3 GPa. In the same range, the temperature dependence of resistivity changes from metallic to semiconducting at 3 GPa and has a plateau region between 50 and 150 K in the semiconducting state. Meanwhile, the pressure variation of band structure shows that the bulk band-gap energy closes at 2.9 GPa and re-opens at higher pressures. Furthermore, according to the Wilson loop analysis, the topological nature of electronic states in noncentrosymmetric BiTeBr at 0 and 5 GPa are explicitly revealed to be trivial and non-trivial, respectively. These results strongly suggest that pressure-induced topological phase transition in BiTeBr occurs at the pressures of 2.9 GPa., 15 pages, 4 figures

The biosynthetic origins of epoxyquinols A (1) and B (2) produced by an unidentified fungus have attracted considerable interest because these compounds could be assembled from a biosynthetic precursor, epoxycyclohexenone aldehyde (3), via an electrocyclization/intermolecular Diels-Alder dimerization cascade reaction. Furthermore, very little is known about the biosynthetic origins of naturally occurring epoxyquinone moieties. We herein describe the incorporation of (13)C at specific positions within the structure of a shunt product, epoxycyclohexenone (4), using stable isotope feeding experiments with sodium [1-(13)C]-acetate and sodium [1,2-(13)C2]-acetate. The results of these experiments strongly suggest that the epoxyquinone skeleton is assembled by a polyketide synthase.

The identification of circulating tumor cells (CTCs) in peripheral blood is a useful approach to estimate prognosis, monitor disease progression and measure treatment effects in several types of malignancies. We have previously used OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. GFP-positive cells (GFP+ cells) were counted under a fluorescence microscope. Our results showed that the number of at least 7.735 µm in diameter GFP+ cells (L-GFP+ cells) in the peripheral blood was a significant marker of prognosis in gastric cancer patients. However, tumor cells undergoing epithelial-mesenchymal transition (EMT) have been reported to be smaller in size than cells without EMT features; thus, CTCs undergoing EMT may escape detection with this technique. Therefore, in this study, we analyzed the relationship between patient outcome and the number of GFP+ cells of any size. We obtained peripheral blood samples from 65 patients with gastric cancer. After infection of OBP-401, GFP+ cells were counted and measured. The relationship between the number of GFP+ cells and surgical outcome was analyzed. The median follow-up period of the surviving patients was 36 months. A significant difference in overall survival was found between patients with 0-5 and patients with ≥6 L-GFP+ cells. No clear relationship was established between the number of small-sized GFP+ cells and patient prognosis. The number of L-GFP+ cells was significantly related to overall survival in patients with gastric cancer. The detection of L-GFP+ cells using OBP-401 may be a useful prognostic marker in gastric cancer.

A putative 7-dimethylallyl tryptophan synthase (DMATS) gene from a fungal Neosartorya sp. was cloned and overexpressed as a soluble His 6 -fusion protein in Escherichia coli. The enzyme was found to catalyze the prenylation of l -tryptophan at the C7 position of the indole moiety in the presence of dimethylallyl diphosphate; thus, it functions as a 7-DMATS. In this study, we describe the biochemical characterization of 7-DMATS from Neosartorya sp., referred to as 7-DMATS Neo , and the structural basis of the regioselective prenylation of l -tryptophan at the C7 position by comparison of the three-dimensional structural models of 7-DMATS Neo with FgaPT2 (4-DMATS) from Aspergillus fumigatus .

Derivatization of a 5′-(vinylsulfonylaminodeoxy)adenosine scaffold with a clickable functionality provided an activity-based probe that was used to label native carrier protein (CP) motifs in nonribosomal peptide synthetases (NRPSs). When coupled with a fluorescent tag, this probe selectively targeted phosphopantetheinylated CPs (holo-form) from recombinant NRPS enzyme systems and in whole proteomes.

Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as "site-directed chemical mutation." In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be noncovalently and nongenetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, β-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of β-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the β-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of Arg

A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.

A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.

Derived from the genus maple (Acer), acertannins are a group of gallotannins which have a characteristic 1,5-anhydro- d -glucitol (1,5-AG) occupying the central core position in the tannic acid structure whose hydroxyl groups have one or more galloyl residues. We have synthesized all ten naturally-occurring acertannins and seven new acertannin derivatives from 1,5-AG. Side-by-side comparisons revealed that 2,4,6-tri- O -galloyl-1,5-AG 21 (maplexin E) and maplexin F 22 (2,3,6-tri- O -galloyl-1,5-AG) were good inhibitors of ceramidase (CDase). In contrast, the core anhydrosugar 1,5-AG 12 itself and 3′,4′,5′-trimethoxy benzoyl derivatives 23 – 25 did not show CDase inhibition. Metabolic labelling experiments using NBD-hexanoic acid revealed that 50 μM of 6- O -galloyl-1,5-AG 16 (Ginnalin B), 2,6-di- O -galloyl-1,5-AG 19 (Ginnalin A), and 4,6-di- O -galloyl-1,5-AG 4 increased intracellular NBD-labeled ceramide, by 2.3, 2.2, and 2.1-fold, respectively. It is noteworthy that these acertannins 16 , 19 , and 4 promoted ceramide synthase 3 (CERS3) gene expression. Acertannins, therefore, represent a new class of potential intracellular ceramide regulators exhibiting both CDase inhibition and ceramide synthase promotion.

Anchorage loss elicits a set of responses in cells, such as transcriptional changes, in order to prevent inappropriate cell growth in ectopic environments. However, the mechanisms underlying these responses are poorly understood. In this study, we investigated the transcriptional up-regulation of cyclin-dependent kinase inhibitor p21(Cip1) during anchorage loss, which is important for cell cycle arrest of nonadherent cells in the G1 phase. Up-regulation was mediated by an upstream element, designated as the detachment-responsive element (DRE), that contained Kruppel-like factor 4 (KLF4) and runt-related transcription factor 1 (RUNX1) recognition sites; both of these together were necessary for transactivation, as individually they were insufficient. RNAi experiments revealed that KLF4 and a multidomain adaptor protein, hydrogen peroxide-inducible clone 5 (HIC-5), were critically involved in DRE transactivation. The role of HIC-5 in this mechanism was to tether KLF4 to DNA sites in response to cellular detachment. In addition, further analysis suggested that oligomerization and subsequent nuclear matrix localization of HIC-5, which was accelerated spontaneously in cells during anchorage loss, was assumed to potentiate the scaffolding function of HIC-5 in the nucleus and consequently regulate p21(Cip1) transcription in a manner responding to anchorage loss. At the RUNX1 site, a LIM-only protein, CRP2, imposed negative regulation on transcription, which appeared to be removed by anchorage loss and contributed to increased transcriptional activity of DRE together with regulation at the KLF4 sites. In conclusion, this study revealed a novel transcriptional mechanism that regulated gene expression in a detachment-dependent manner, thereby contributing to anchorage-dependent cell growth.

We targeted development of a dehydratase (DH) specific reactive probe that can facilitate detection, enrichment, and identification of DH enzymes in fatty acid synthases (FAS) and polyketide synthases (PKS). The first reported mechanism based inactivator, 3-decynoyl-N-acetylcysteamine (3-decynoyl-NAC), while active against the Escherichia coli β-hydroxydecanoyl thiol ester DH FabA, translates poorly to an activity-based probe due to non-specific reactivity of the thioester moiety. Here we describe the design, synthesis and utility of a DH-specific probe that contains a sulfonyl 3-alkyne reactive warhead engineered to avoid hydrolysis or non-enzymatic inactivation. When coupled with a fluorescent tag, this probe targets DH enzymes from recombinant type I and type II FAS and PKS enzyme systems and in whole proteomes. Activity studies, including FabA inactivation and antibiotic susceptibility, suggest that this sulfonyl 3-alkyne scaffold selectively targets a common dehydratase mechanism. These studies indicate that the DH-specific mechanism based probe can greatly accelerate both the functional characterization and molecular identification of virtually any type of FAS and PKS in complex proteomes.

Mitochondria are considered to play an important role in oxidative stress response since they are a source of reactive oxygen species and are also targeted by these species. This study examined the mitochondrial conditions in cells of epithelial origin that were exposed to H(2)O(2) and found a decline in the membrane potential along with a specific loss of UQCRC1, a sub-unit of complex III, suggesting that mitochondrial dysfunction occurs upon exposure to oxidative stress. This observation led to the hypothesis that certain cellular responses to oxidative stress occurred because of mitochondrial dysfunction. When mitochondria-less (pseudo ρ0) cells were examined as a model of mitochondrial dysfunction, striking similarities were found in their cellular responses compared with those found in cells exposed to oxidative stress, including changes in gene expression and gelatinolytic enzyme activities, thus suggesting that cellular responses to oxidative stress were partly mediated by mitochondrial dysfunction. This possibility was further validated by microarray analysis, which suggested that almost one-fourth of the cellular responses to oxidative stress were mediated by mitochondrial dysfunction that accompanies oxidative stress, thereby warranting a therapeutic strategy that targets mitochondria for the treatment of oxidative stress-associated diseases.

We succeeded in synthesizing Ba2RBiO6 (R = La, Ce, Nd, Sm, Eu, Gd, and Dy) with a double-perovskite structure. Its photocatalytic activities were then investigated for a degradation of methylene blue (MB) solution and gaseous 2-propanol (IPA). High photocatalytic activities were observed under visible light irradiation and also a rare earth dependence of photocatalytic activities in the IPA degradation. We carried out first-principle calculations of the band structure based on the all-electron Full-potential Linear Augmented Plane-Wave (FLAPW) method and discuss the relationship between the high photocatalytic activities and the band structure in this paper.

Transforming growth factor (TGF)-beta regulates the expression of matrix metalloproteinases (MMPs) and components of the extracellular matrix, thereby profoundly affecting the microenvironment of cells including cancerous ones. We studied MMP-10 induction by TGF-beta in mammary epithelial cells and found that the induction was dependent on the myocyte enhancer factor (MEF)-2 transcription factor. TGF-beta upregulated the gene promoter through the MEF2 site, and knockdown of the MEF2A transcription factor negatively affected MMP-10 induction, whereas its overexpression had a positive effect on the induction. In response to TGF-beta, acetylation and concomitant binding of MEF2A to the promoter region increased, thus suggesting a critical role of MEF2A in transactivation of MMP-10 by TGF-beta. Consistent with the fact that class IIa histone deacetylases (HDACs) interact with MEF2 and suppress transcription, knockdown of HDACs increased and their overexpression inhibited MMP-10 expression. Intriguingly, TGF-beta promoted proteasome-dependent degradation of HDACs. Consistent with this, acetylation of core histones was increased around the MEF2 site of the MMP-10 promoter by TGF-beta and alleviated by overexpression of HDACs. Collectively, it is possible that TGF-beta transcriptionally upregulated MMP-10 through activation of MEF2A, concomitant with acetylation of core histones increasing around the promoter, as a consequence of degradation of the class IIa HDACs.

Structural change in Bi2Te3 under high pressure up to 16.6 GPa has been studied by powder x-ray diffraction. We observed two times of phase transitions at room temperature at the pressures of 8 and 14 GPa, respectively. According to our preliminary result on electrical resistance, it is reasonable to suppose that superconducting transition with T c =2.8 K at the pressures of 10.2 GPa is observed in phase II. On the other hand, we found anomalies of the pressure dependences of lattice parameters and volume at around 2 GPa, which probably means the change in electrical structure on the Fermi surface. †This paper was presented at the XLVIth European High Pressure Research Group (EHPRG 46) Meeting, Valencia (Spain), 7–12 September, 2008.

Mitochondrial dysfunction, in particular, interference in the respiratory chain, is often responsible for the toxicogenic effects of xenobiotics. In this study, changes in gene expression resulting from pharmacological inhibition of the respiratory chain were studied by DNA microarray analysis using cells treated with rotenone or antimycin A, which inhibit complexes I and III of the electron transport system, respectively. Forty-eight genes were either up- or down-regulated more than 3-fold. These included stress- and/or metabolic-related effector genes and several transcriptional regulators represented by CHOP-10. Further study using siRNA showed that among the four genes studied, up-regulation of three was dependent on CHOP-10. C/EBPbeta, a dimerizing partner of CHOP-10, was also involved in two of the three genes including Trib3, implying that CHOP-10, heterodimerizing with C/EBPbeta or another partner played a key role in the expression of a set of genes under stress. Although CHOP-10 and Trib3 were both ER-stress response genes, signal inducing Trib3 during mitochondrial stress was distinct from that during ER stress. Cytotoxicity caused by inhibition of the respiratory chain was attenuated by treatment with siRNA for CHOP-10. This study demonstrated the importance of CHOP-10 in coordinating individual gene expression in response to the mitochondrial stress.

Anchorage dependence of cell growth and survival is a critical trait that distinguishes nontransformed cells from transformed cells. We demonstrate that anchorage dependence is determined by anchorage-dependent nuclear retention of cyclin D1, which is regulated by the focal adhesion protein, Hic-5, whose CRM1-dependent nuclear export counteracts that of cyclin D1. An adaptor protein, PINCH, interacts with cyclin D1 and Hic-5 and potentially serves as an interface for the competition between cyclin D1 and Hic-5 for CRM1. In nonadherent cells, the nuclear export of Hic-5, which is redox-sensitive, was interrupted due to elevated production of reactive oxygen species, and cyclin D1 was exported from the nucleus. When an Hic-5 mutant that was continuously exported in a reactive oxygen species-insensitive manner was introduced into the cells, cyclin D1 was retained in the nucleus under nonadherent conditions, and a significant population of cells escaped from growth arrest or apoptosis. Interestingly, activated ras achieved predominant cyclin D1 nuclear localization and thus, growth in nonadherent cells. We report a failsafe system for anchorage dependence of cell growth and survival.

We found that a specific isoform of hepatocyte nuclear factor 4alpha (HNF-4alpha), HNF-4alpha8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-beta. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4alpha8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4alpha8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4alpha8 in TGF-beta signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-beta1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-beta1 was attenuated by forced expression of HNF-4alpha8. HMGA2 has recently emerged as a transcriptional organizer of TGF-beta signaling, regulating several key factors involved in epithelial-mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4alpha as a new target downstream of HMGA2 and assigned a new role to HNF-4alpha in the TGF-beta signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells.

Jumping translocation breakpoint (JTB) is a gene located on human chromosome 1 at q21 that suffers an unbalanced translocation in various types of cancers, and potentially encodes a transmembrane protein of unknown function. The results of cancer profiling indicated that its expression was suppressed in many cancers from different organs, implying a role in the neoplastic transformation of cells. Recently, we isolated JTB as a TGF-beta1-inducible clone by differential screening. In this study, we characterized its product and biological functions. We found that it was processed at the N-terminus and located mostly in mitochondria. When expressed in cells, JTB-induced clustering of mitochondria around the nuclear periphery and swelling of each mitochondrion. In those mitochondria, membrane potential, as monitored with a JC-1 probe, was significantly reduced. Coinciding with these changes in mitochondria, JTB retarded the growth of the cells and conferred resistance to TGF-beta1-induced apoptosis. These activities were dependent on the N-terminal processing and induced by wild-type JTB but not by a mutant resistant to cleavage. These findings raised the possibility that aberration of JTB in structure or expression induced neoplastic changes in cells through dysfunction of mitochondria leading to deregulated cell growth and/or death.

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs.

Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments.

We describe competitive activity-based protein profiling (ABPP) to accelerate the functional prediction and assessment of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) in proteomic environments. Using a library of sulfamoyloxy-linked aminoacyl-AMP analogs, the competitive ABPP technique offers a simple and rapid assay system for adenylating enzymes and provides insight into enzyme substrate candidates and enzyme active-site architecture.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large diverse families of biosynthetic enzymes that catalyze the synthesis of natural products that display biologically important activities. Genetic investigations have greatly contributed to our understanding of these biosynthetic enzymes; however, proteomic studies are limited. Here we describe the application of active site-directed proteomic probes for adenylation (A) domains to profile the activity of NRPSs directly in native proteomic environments. Derivatization of a 5'-O-N-(aminoacyl)sulfamoyladenosine appended clickable benzophenone functionality enabled activity-based protein profiling of the A-domains in NRPSs in proteomic extracts. These probes were used to identify natural product producing microorganisms, optimize culture conditions, and profile the activity dynamics of NRPSs. Our proteomic approach offers a simple and versatile method to monitor NRPS expression at the protein level and will facilitate the identification of orphan enzymatic pathways involved in secondary metabolite production.

The field induced magnetic phase transitions of Cs 2 CuBr 4 were investigated by means of magnetization process and neutron scattering experiments. Cs 2 CuBr 4 should be characterized as S = 1/2 two-dimensional triangular antiferromagnet. Below the ordering temperature T N = 1.4 K, the Spin structure is the helical incommensurate structure almost within the triangular lattice plane. In the field direction within the triangular lattice plane, Cs 2 CuBr 4 exhibits the magnetization plateaux at one-third and two-thirds of the saturation magnetization. The spin structure in the one-third plateau phase is found to be almost collinear up-up-down structure which should be stabilized by quantum fluctuation as predicted by the theoretical studies.

The structural phase transition induced by magnetic fields on MnAs was observed by the X-ray diffraction measurements in high magnetic fields up to 4 T . Magnetization measurements showed that Curie temperature TC was 315.5 K for increasing temperatures and a metamagnetic transition from the paramagnetic to the ferromagnetic state occurred above TC. The structural transformation from the hexagonal NiAs-type to the orthorhombic MnP-type structure was confirmed at TC with increasing temperature from 290 to 319 K via two-phase coexistence region. The X-ray diffraction profiles at 319 K showed the single phase of the MnP-type structure in zero field and applying magnetic field of 3 T caused appearance of the Brag peak of the hexagonal structure. On further increase of magnetic fields, the single phase of the hexagonal structure was observed above 3.5 T in the forced ferromagnetic state. Both the magnetic and structural transitions induced by magnetic fields above TC were first order with a hysteresis and had a close relationship between each other.

Structural transformation induced by magnetic fields on MnAs and MnAs 0 . 9 Sb 0 . 1 was investigated by the X-ray diffraction measurements in high magnetic fields up to 5 T. The X-ray diffraction profiles at 319K for MnAsshowed a single phase of the orthorhombic MnP-type structure in zero field, and applying a magnetic field of 3 T caused an appearance of the hexagonal NiAs-type structure. On further increase of magnetic fields, the single phase with the hexagonal structure was confirmed above 3.5 T in a forced ferromagnetic state. The X-ray diffraction profiles at 295 K for MnAs 0 . 9 Sb 0 . 1 showed the hexagonal NiAs-type structure. However, the coexistence of two kinds of the NiAs-type structure with different lattice parameters was confirmed in the magnetic field of 2.5 T. The two phase coexistence was also confirmed in the temperature variation measurements in zero magnetic field. The lattice volume of the ferromagnetic phase was 1.1% larger than that of the paramagnetic phase. These results imply that the transition at T C for MnAs 0 . 9 Sb 0 . 1 is of the first-order.

To investigate the valence transition of Eu in the pseudo-binary compounds series, EuCu 2 (Si x Ge 1- x ) 2 , measurements of the lattice constants, the X-ray absorption spectroscopy (XAS), the mag...

The magnetic and structural influences of hydrogen absorption were studied on the itinerant electron metamagnet YCo 2 . The system YCo 2 H x forms two crystalline hydrides of the α phase (0 x x ≤3.5). The hydride samples also include small amounts of the non-intrinsic ferromagnetic component due to the partial structural disordering upon hydrogen absorption. The high field susceptibility indicated that the α phase is an itinerant electron metamagnet in which the critical field is possibly lower than that of YCo 2 . The β phase has no metamagnetic transition. The hydrogen-induced changes in the magnetic properties are discussed on the basis of the itinerant electron magnetism.

The magnetization processes of the Y(Co 1 −x Ni x ) 5 system were measured at 4.2 K in pulsed high magnetic fields up to 40 T. The spontaneous magnetization decreases linearly with increasing Ni concentration up to x =0.4. In the narrow concentration region 0.5⩽ x ⩽0.6, the magnetization rapidly decreases, but weak ferromagnetism persists up to x =0.8. On the other hand, the high-field susceptibility exhibits a maximum at x =0.55. These results show that the magnetic state of Y(Co 1− x Ni x ) 5 changes from the high- to the low-moment state at the critical concentration x =0.55. In the vicinity of this concentration, the magnetization was measured at high pressures up to 1 GPa. The compound Y(Co 0 . 5 Ni 0 . 5 ) 5 shows a very large pressure effect, indicating that it is very close to the critical concentration.

The effect of alloying on the first-order valence transition in Yb 1− x R x InCu 4 (R=Y, La, Ce, Lu) has been studied by magnetization measurements. It has been found that the transition temperature T v increases by substitution of La and Ce for Yb and decreases for the Y and Lu substitutions. In order to reveal the volume contribution to the change of T v , we measured lattice parameters of Yb 1− x R x InCu 4 at room temperature. The lattice parameter increases with x for R=Ce, La, Y and slightly decreases for R=Lu. Using the data of compressibility and the pressure effect on T v for YbInCu 4 we found that the changes of T v by La and Ce alloying are consistent with the volume change. However, both Y and Lu substitutions for Yb lead to a decrease in T v , inconsistent with the volume change. Possible reasons for the decrease of the Kondo temperature below T v by Y and Lu alloying are discussed.

We synthesized the powder samples of KMgxCu5−xV3O13 (x=0.1∼1) and investigated their magnetic susceptibilities and the magnetization curves up to 47 T. The Curie constant and the Weiss temperature of KMgCu4V3O13 change markedly around 20 K. From the Curie constant estimated from the data between 5 and 10 K, the number of spins in the low-temperature region was estimated to be nlow=8.13%, assuming all the copper spins have S=1/2 and g=2. The magnetization curve of KMgCu4V3O13 at 1.7 K is bent around 20 T and shows a linear increase between 20 and 30 T. The magnetization, Mlinear(H→0), estimated by extrapolating the linear part to 0 T is 0.306μB/f.u. Furthermore, all the x dependences of the Neel temperature TN, nlow, θlow, the magnetic field at spin–flop transition Hsf and Mlinear(H→0) change continuously with x. This indicates that there is no quantum critical region between the frustrated spin chains of KCu5V3O13 and KMgCu4V3O13. The ground states of KCu5V3O13 and KMgCu4V3O13 have been deduced.

The magnetic field effects on the thermodynamic properties of chemical reactions were systematically studied. In the formulation, the chemical influence of magnetic fields due to magnetostatic energy was represented by the magneto-chemical potential for each component and the magnetic free energy change per unit reaction. Then the general formulae were derived for the magnetic field-induced changes in the equilibrium constant and the heat, free energy and entropy of reaction. In the calculation, the general formulae were applied to a ferromagnetic metal-hydrogen reaction and the magnetic field effects were numerically evaluated for two reactions in the single system LaCo5–H2. In the experiment, the magnetic field effects were observed by applying high magnetic fields of 10–30 T to the system LaCo5–H2. The observed results agreed qualitatively and quantitatively with the corresponding calculated ones. This study resolved the issue of the thermodynamic effects of magnetic fields on chemical reactions.

We experimentally investigated the effects of magnetic fields on the heat of reaction by examining the metal–hydrogen system LaCo 5 –H 2 . The heat of reaction was measured in zero field and a magnetic field by the logarithmic pressure vs. inverse temperature method and the calorimetric one. Both the methods gave generally the same results that the magnetic field caused the absolute value of the heat of reaction to increase a little in the α+β region and decrease considerably in the β+γ region. These agree well with the calculation based on the general formulation of magneto-thermodynamic effects in chemical reactions. This is the first observation of the magnetic field effect on the heat of reaction.

The magnetic properties of LaCo 5 hydrides and deuterides were investigated by (1) in situ magnetization at 77 K in 20 T which related magnetization with hydrogen/deuterium composition, and (2) high field magnetization at 4.2 K up to 44 T for well-characterized samples. The β-phase LaCo 5 H(D) 3.4 is recognized as a saturated ferromagnet, like the metal phase LaCo 5 . The γ-phase LaCo 5 H(D) 4.3 undergoes a metamagnetic transition from a low moment state to a high moment state at a critical field of 35 T (hydride) or 38 T (deuteride). These magnetic properties are discussed in relation to the electronic structure.

The electromotive force (emf) has been investigated for the hydride–hydride electrochemical cell under the influence of gradient magnetic fields. The thermodynamic consideration presumed the relationships between the emf and the gradient between the electrodes. The emf was measured for the array of the electrodes using the ferromagnetic metal hydride LaCo 5 H x in the magnetic fields with the maximum gradient of 106 T/m up to 15 T at 293 K. The emf was proportional to the averaged gradient of magnetic fields and the distance between two electrodes and depended on the magnetic properties of the electrode material, which agreed well with the thermodynamic consideration.

One-step conversion of various types of formate esters into the corresponding O-silyl ethers under neutral reaction conditions was established by employing samarium diiodide in the presence of chlorosilane reagents.