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Background/aim: Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and has been approved for the treatment of non-small cell lung cancers (NSCLCs) with EGFR mutations. Here we demonstrated that gefitinib induced a significantly enhanced biological activity of succinate-tetrazolium reductase (STR) in mitochondria and mitochondrial membrane potential in HCC827 cells (EGFR mutation NSCLCs, sensitive to gefitinib) at a high cell density., Materials and Methods: We assessed the biological activity (STR, mitochondrial membrane potential, expression level of Bcl-2 family proteins) of gefitinib on NSCLCs at different cell densities., Results: The 3D cell culture experiments showed the enhanced mitochondrial biological activity in clustered cell culture treated with gefitinib. Interestingly, the expression levels of Bcl-x L and Bax, were affected by the cellular number and gefitinib treatment. We also found that gefitinib prevented additive anticancer activity in the combinational treatment with doxorubicin, which induces mitochondria-dependent apoptotic cell death., Conclusion: Our results indicate that gefitinib may work as a mitochondrial protector against combinational treatment with mitochondria-dependent anticancer agents in high-cell-density., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Extracellular vesicles (EVs) including exosomes have been shown to play crucial roles in cell-to-cell communication because of their ability to carry biofunctional molecules (e.g., microRNAs and enzymes). EVs also have pharmaceutical advantages and are highly anticipated to be a next-generation intracellular delivery tool. Here, we demonstrate an experimental technique that uses arginine-rich cell-penetrating peptide (CPP)-modified EVs to induce active macropinocytosis for effective cellular EV uptake. Modification of arginine-rich CPPs on the EV membrane resulted in the activation of the macropinocytosis pathway, and the number of arginine residues in the peptide sequences affected the cellular EV uptake efficiency. Consequently, the ribosome-inactivating protein saporin-encapsulated EVs modified with hexadeca-arginine (R16) peptide effectively attained anti-cancer activity.

We demonstrate a novel system for inducing clustering of cell surface receptors via recognition peptide segments displayed on exosomes, leading to receptor activation. With this system, targeting of receptor-expressing cells and facilitation of the endocytic uptake of exosomes, which contained the anti-cancer protein saporin, were successfully achieved, leading to cell death.

Conformationally constrained peptides have been developed as an inhibitor for protein-protein interactions (PPIs), and we have de novo designed cyclized helix-loop-helix (cHLH) peptide with a disulfide bond consisting of 40 amino acids to generate molecular-targeting peptides. However, synthesis of long peptides has sometimes resulted in low yield according to the respective amino acid sequences. Here we developed a method for efficient synthesis and labeling for cHLH peptides. First, we synthesized two peptide fragments and connected them by the copper-mediated alkyne and azide cycloaddition (CuAAC) reaction. Cyclization was performed by bis-thioether linkage using 1,3-dibromomethyl-5-propargyloxybenzene, and subsequently, the cHLH peptide was labeled with an azide-labeled probe. Finally, we designed and synthesized a peptide inhibitor for the p53-HDM2 interaction using a structure-guided design and successfully labeled it with a fluorescent probe or a functional peptide, respectively, by click chemistry. This macrocyclization and labeling method for cHLH peptide would facilitate the discovery of de novo bioactive ligands and therapeutic leads. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 415-421, 2016., (© 2016 Wiley Periodicals, Inc.)

Previously, we reported that MyoD, a master gene for myogenic cells, could efficiently convert primary skin fibroblasts into myoblasts and myotubes, thereby effecting direct reprogramming. In this study, we further demonstrated that MyoD-expressing primary fibroblasts displayed rapid movement in culture, with a movement velocity that was significantly faster, almost four times, than mouse primary myoblasts. MyoD-transduced cells obtained the characteristics of Ca 2 + release and electrically-stimulated contraction, which was comparable to C2C12 myotubes, suggesting that the essential features of muscle were observed in the transduced cells. Furthermore, the ability to fuse to the host myoblasts means that gene transfer from MyoD-transduced cells to host muscle cells could be obtained by cell fusion. In comparison with the iPS method (indirect reprogramming), our transduction method has a low risk for tumorigenesis and carcinogenesis because the starting cells are fibroblasts and the transduced cells are myoblasts, both normal and mortal cells. Accordingly, MyoD transduction of human skin fibroblasts using the adenoviral vector is a simple, inexpensive and promising candidate as a new cell transplantation therapy for patients with muscular disorders.

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 H52 in 27C1 makes a hydrogen bond with the P═O oxygen in the hapten. This suggested that during β-elimination, Arg H52 of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the β-elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [(k cat /K m 2)/k uncat ] of 1 000 000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the electrostatic interactions. In 27C1, the cationic guanidyl group of Arg H52 is spatially close to the nitrogen of the amine components. In this microenvironment, the intrinsic pK a of the amine is perturbed and shifts to a lower pK a , which efficiently abstracts the α-proton during the reaction. This mechanism is consistent with the observed kinetic isotope effect (E2 or E1cB mechanism). Thus, site-directed chemical mutation provides a better understanding of enzyme functions and opens new avenues in biocatalyst research.

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.

In recent years, interest in biological activities of compounds from marine organisms has intensified. Cancer is the most principal enemy for human life and health. For the first time, to the best of our knowledge, we investigated a novel algae-derived polysaccharide for its role in inducing apoptosis and cell cycle arrest in human gastric carcinoma MKN45 cells. We found that the novel polysaccharide suppressed MKN45 cell proliferation, induced cell apoptosis and arrested the cells at G2/M phase. Furthermore, we observed that the generation of reactive oxygen species (ROS) and the phosphorylation of Jun N-terminal kinase (JNK), p53, caspase-9 and -3 were induced in the polysaccharide-treated MKN45 cells. In addition, pretreatment with N-acetyl-cysteine (NAC) and SP600125, the inhibitor of ROS and JNK, induced MKN45 cell proliferation, prevented the cell apoptosis and released the cells from cycle arrest. Finally, we found that pretreatment with NAC prevented the JNK, p53, caspase-9 and -3 protein phosphorylation induced by the polysaccharide, however, pretreatment with SP600125 did not affect the generation of ROS, suggesting that ROS is upstream of JNK. Taken together, the novel polysaccharide induced cancer cell apoptosis and arrested cell cycle via ROS/JNK signaling pathway.

The design of inhibitors of intracellular protein-protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix-loop-helix (cHLH) peptide as a scaffold for generating cell-permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell-permeability. To inhibit p53-HDM2 interactions, the p53 epitope was grafted onto the C-terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53-R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53-R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well-structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We analyzed the correlation between the conformational strain and the binding kinetics in antigen-antibody interactions. The catalytic antibodies 6D9, 9C10, and 7C8 catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. The crystal structure of 6D9 complexed with a transition-state analog (TSA) suggests that 6D9 binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable twisted conformation, enabling the substrate to reach the transition state during catalysis. The present binding kinetic analysis showed that the association rate for 6D9 binding to the substrate was much lower than that to TSA, whereas those for 9C10 and 7C8 binding were similar to those to TSA. Considering that 7C8 binds to the substrate with little conformational change in the substrate, the slow association rate observed in 6D9 could be attributed to the conformational strain in the substrate.

Heterologous expression of four candidate genes found in ophiobolin gene clusters from three fungal strains was employed to elucidate the late-stage biosynthetic pathway of phytotoxin ophiobolin. Expression of oblBAc (cytochrome P450) from the cryptic gene cluster gave unexpected products, and that of oblBBm/oblBEv from the gene cluster of ophiobolin producers, with oblDBm as the transporter, yielded intermediate ophiobolin C through an unusual four-step oxidation process. The observation made in this study may provide a useful guideline for the elucidation of genuine biosynthetic pathways of natural products.

Reconstitution of whole biosynthetic genes in Aspergillus oryzae has successfully applied for total biosynthesis of various fungal natural products. Heterologous production of fungal metabolites sometimes suffers unexpected side reactions by host enzymes. In the studies on fungal polyketides solanapyrone and cytochalasin, unexpected oxidations of terminal olefin of biosynthetic intermediates were found to give one and four by-products by host enzymes of the transformants harboring biosynthetic genes. In this paper, we reported structure determination of by-products and described a simple solution to avoid the undesired reaction by introducing the downstream gene in the heterologous production of solanapyrone C.

Non-reducing iterative type I polyketide synthase genes, pnk1 and pnk2, were cloned from the fungus Phoma sp. BAUA2861, which produces the topoisomerase I inhibitors, topopyrones A to D. Heterologous expression of these polyketide synthase genes under the α-amylase promoter in Aspergillus oryzae was carried out to identify their functions. The pnk2 transformant produced topopyrones C, D, and haematommone. Therefore, the pnk2 gene was found to encode for the topopyrone nonaketide synthase.

The characteristics of 2 Glycyrrhiza plants, G. glabra and G. bucharica (=Meristotropis bucharica), were investigated in Tajikistan. The glycyrrhizin content in the underground parts of G. glabra varied from 2.56 to 9.29% of the dry weight, and the content of glabridin, a species-specific flavonoid of G. glabra, varied from 0.09 to 0.92% of the dry weight. Seeds of G. glabra plants from Tajikistan were cultivated for 3 years in Japan, and the glycyrrhizin content of the harvested roots ranged from 0.75 to 1.82% of the dry weight. In addition, HPLC analysis of leaf extracts indicated that the G. glabra plants collected in Tajikistan could be divided into various types, according to the flavonoid contents of the leaves. The endemic G. bucharica was also collected. A phylogenetic tree of rbcL nucleotide sequences from various Glycyrrhiza plants indicated that G. bucharica was closely related to the three glycyrrhizin-producing Glycyrrhiza spp. (G. uralensis, G. inflata, and G. glabra), even though G. bucharica does not produce glycyrrhizin.

In the title racemic compound, C14H15NO, the hy-droxy and amino groups form a bent tweezer-like motif towards the phenyl groups. In the crystal, enanti-omers aggregate with each other and are linked by O-H⋯N hydrogen bonds, forming chiral 21-helical columnar structures from C(5) chains along the b-axis direction. Left- and right-handed 21 helices are formed from (1S,2R)-2-amino-1,2-di-phenyl-ethanol and (1R,2S)-2-amino-1,2-di-phenyl-ethanol, respectively.

The title compound, C5H9NO4, crystallized as a zwitterion. There is an intra-molecular N-H⋯O hydrogen bond involving the trans-succinic acid and the ammonium group, forming an S(6) ring motif. In the crystal, mol-ecules are linked by O-H⋯O hydrogen bonds, forming C(7) chains along the c-axis direction. The chains are linked by N-H⋯O and C-H⋯O hydrogen bonds, forming sheets parallel to the bc plane. Further N-H⋯O hydrogen bonds link the sheets to form a three-dimensional framework.

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

The blood concentration of intact proinsulin, but not total proinsulin, has been suggested to be a diagnostic marker for type 2 diabetes mellitus (T2DM), but a sensitive assay specific for rodent intact proinsulin is lacking. Here, a novel enzyme-linked immunosorbent assay (ELISA) for mouse intact proinsulin was developed. The developed ELISA detected mouse intact proinsulin with the working range of 8.3 to 2700pg/ml. Cross-reactivity with mouse split-32,33 proinsulin was approximately 100times lower than the reactivity with mouse intact proinsulin, and no cross-reactivity with mouse insulin was detected. The developed ELISA was sufficiently sensitive to detect low levels of intact proinsulin in normal mouse plasma. The measurement by the developed ELISA revealed that intact proinsulin was elevated in the plasma of type 2 diabetic db/db mice as mice aged, and the ratio of intact proinsulin/insulin in plasma was correlated with levels of glycated hemoglobin A1c as seen in T2DM patients. These results suggest that the plasma level of intact proinsulin, but not total proinsulin, is a sensitive marker for pancreatic dysfunction and the ensuring diabetic disease progression of db/db mice. This ELISA could aid nonclinical evaluation of therapeutic interventions in T2DM., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Talaromyces stipitatus ATCC 10500 possesses 17 non-reducing polyketide synthase (NR-PKS) genes. During the course of our functional analysis of PKS genes with a C-terminus methyltransferase domain from T. stipitatus, we expressed tspks2, tspks3 and tspks4 genes in the heterologous host Aspergillus oryzae, respectively. Although the tspks4 transformant gave no apparent product in HPLC analysis, a novel azaphilone pentaketide (3) was identified along with two known related products from the tspks2 transformant. Of four hexaketide products from the tspks3 transformant, two new compounds were identified to be 2-acetyl-7-methyl-3,6,8-trihydroxynaphthalene (4) and its derivative fused with α-methyl-α,β-unsaturated γ-lactone (7)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes., (Copyright © 2015. Published by Elsevier Inc.)

The acylalkylpyrone synthase CsyB from Aspergillus oryzae catalyzes the one-pot formation of the 3-acyl-4-hydroxy-6-alkyl-α-pyrone scaffold from acetoacetyl-CoA, fatty acyl-CoA, and malonyl-CoA. This is the first type III polyketide synthase that performs not only the polyketide chain elongation but also the condensation of two β-ketoacyl units. The crystal structures of wild-type CsyB and its I375F and I375W mutants were solved at 1.7-, 2.3-, and 2.0-Å resolutions, respectively. The crystal structures revealed a unique active site architecture featuring a hitherto unidentified novel pocket for accommodation of the acetoacetyl-CoA starter in addition to the conventional elongation/cyclization pocket with the Cys-His-Asn catalytic triad and the long hydrophobic tunnel for binding the fatty acyl chain. The structures also indicated the presence of a putative nucleophilic water molecule activated by the hydrogen bond networks with His-377 and Cys-155 at the active site center. Furthermore, an in vitro enzyme reaction confirmed that the (18)O atom of the H2(18)O molecule is enzymatically incorporated into the final product. These observations suggested that the enzyme reaction is initiated by the loading of acetoacetyl-CoA onto Cys-155, and subsequent thioester bond cleavage by the nucleophilic water generates the β-keto acid intermediate, which is placed within the novel pocket. The second β-ketoacyl unit is then produced by polyketide chain elongation of fatty acyl-CoA with one molecule of malonyl-CoA, and the condensation with the β-keto acid generates the final products. Indeed, steric modulation of the novel pocket by the structure-based I375F and I375W mutations resulted in altered specificities for the chain lengths of the substrates., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

BTK has emerged as a promising target for treating B-cell malignancies and autoimmune diseases, and there has been a growing demand to identify selective BTK inhibitors efficiently. In this Letter, we have designed and synthesized a new fluorescent probe to screen compounds that preferentially bind to an unactivated state of BTK (BTK [U]). The fluorescence of the probe was turned on in the presence of BTK [U], and quenched by the addition of compounds which preferentially bind to BTK [U]. This unique fluorescent probe was successfully applied to the screening of a kinase focused compound library. The results suggest that this new method is a simple and easy-to-perform assay to screen inhibitors of BTK [U]., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Inflammasome activation initiates the development of many inflammatory diseases, including obesity and type 2 diabetes. Therefore, agents that target discrete activation steps could represent very important drugs. We reported previously that ILG, a chalcone from Glycyrrhiza uralensis, inhibits LPS-induced NF-κB activation. Here, we show that ILG potently inhibits the activation of NLRP3 inflammasome, and the effect is independent of its inhibitory potency on TLR4. The inhibitory effect of ILG was stronger than that of parthenolide, a known inhibitor of the NLRP3 inflammasome. GL, a triterpenoid from G. uralensis, had similar inhibitory effects on NLRP3 activity, but high concentrations of GL were required. In contrast, activation of the AIM2 inflammasome was inhibited by GL but not by ILG. Moreover, GL inhibited NLRP3- and AIM2-activated ASC oligomerization, whereas ILG inhibited NLRP3-activated ASC oligomerization. Low concentrations of ILG were highly effective in IAPP-induced IL-1β production compared with the sulfonylurea drug glyburide. In vivo analyses revealed that ILG potently attenuated HFD-induced obesity, hypercholesterolemia, and insulin resistance. Furthermore, ILG treatment improved HFD-induced macrovesicular steatosis in the liver. Finally, ILG markedly inhibited diet-induced adipose tissue inflammation and IL-1β and caspase-1 production in white adipose tissue in ex vivo culture. These results suggest that ILG is a potential drug target for treatment of NLRP3 inflammasome-associated inflammatory diseases., (© 2014 Society for Leukocyte Biology.)

This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two β-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs.

The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C9-C17 in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

CsyB from Aspergillus oryzae is a novel type III polyketide synthase that catalyzes the formation of csypyrone B1 [4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid] from fatty acyl-CoA, malonyl-CoA and acetoacetyl-CoA. Recombinant CsyB expressed in Escherichia coli was crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space P2₁, with unit-cell parameters a=70.0, b=104.8, c=73.5 Å, β=114.4°.

The development of immunoadsorbents that have high specificity for immunoglobulin and no immunogenicity is essential for immunoadsorption treatment of autoimmune diseases. In this study, we designed peptide immunoadsorbents by molecular grafting of the IgG-Fc binding epitopes of Protein A onto a de novo-designed helix-loop-helix peptide. Linear (linG7A5) and cyclic (cyG7A5) grafted peptides were synthesized to test their binding affinity and specificity. Peptide cyG7A5 demonstrated high specificity for human IgG-Fc, with a K(D) of 19 μM, and demonstrated no affinity to other plasma proteins, human serum albumin, or fibrinogen. To evaluate their immunoadsorbance efficiency, the grafted peptides and Protein A were conjugated to polyvinyl acetate resin and tested in a batch-wise process for adsorption removal of IgG from human plasma. The IgG capture capacities of the peptides correlated well with their binding affinities. Interestingly, cyG7A5 showed a higher binding specificity for IgG than did Protein A., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening.

Catalytic antibody 27C1 bears binding sites for both a substrate- and a functionalized small nonprotein component in the active site. We investigated the possibility of exploiting imine and enamine intermediates using a primary amine molecule into the active site of antibody 27C1. The antibody catalyzed β-keto acid decarboxylation with a rate enhancement (kcat/Km/kuncat) of 140,000, as well as highly regioselective cross-aldol reactions of ketones and p-nitrobenzaldehyde. These studies provide new strategies for the generation of catalytic antibodies possessing binding sites for functionalized components., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Csypyrones B1, B2 and B3 are α-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-(13)C] and [2-(13)C] acetate feeding experiments. (13)C NMR analyses of the methyl esters of the csypyrone B compounds fed with the (13)C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Epigenetic manipulation of gene expression in Chaetomium indicum using a HDAC inhibitor led to the isolation of structurally diverse chaetophenols, and 3, 4 and 5 bear unprecedented polycyclic skeletons. The expression of two silent genes (pksCH-1 and pksCH-2) for nonreducing PKSs involved in chaetophenol biosynthesis was associated with an increase of histone acetylation level. The heterologous gene expression study in Aspergillus oryzae revealed pksCH-2 to be the NR-PKS gene for 8.

Since our first report on the identification of the fungal type III polyketide synthase (PKS) genes csyA~D in Aspergillus oryzae RIB40, type III PKS homologues have also been found in other fungal species. We previously reported the isolation and structural determination of csypyrone B1 as the main product of CsyB when inductively expressed in Aspergillus oryzae. Herein we report the isolation and identification of the two minor products of the csyB transformant in addition to csypyrone B1 as 4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid and 5-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)pentanoic acid. These compounds were named csypyrone B2 and B3, respectively, and both are homologues of main product csypyrone B1 with different side chain lengths. This result suggests that the carbon skeleton of the csypyrone B precursor is constructed by the condensation of fatty acyl-CoA and acetylmalonyl-CoA followed by pyrone formation. The alkyl side chain of the precursor may be oxidatively cleaved by enzyme(s) in the host fungus to give variations of csypyrone B with propanoic acid, butyric acid, or pentanoic acid side chains., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

RamR is a transcriptional repressor of the gene-encoding RamA protein, which controls the expression of the multidrug efflux system genes acrAB-tolC. RamR is an important multidrug-resistance factor, however, its structure and the identity of the molecules to which it responds have been unknown. Here, we report the crystal structure of RamR in complex with multiple drugs, including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. All compounds are found to interact with Phe155 of RamR, and each compound is surrounded by different amino acid residues. Binding of these compounds to RamR reduces its DNA-binding affinity, which results in the increased expression of ramA. Our results reveal significant flexibility in the substrate-recognition region of RamR, which regulates the bacterial efflux participating in multidrug resistance.

A triterpene saponin, glucoglycyrrhizin, was isolated from a glycyrrhizin-deficient strain 83-555 of Glycyrrhiza uralensis (Leguminosae), and the structure was determined by chemical and spectral data to be 3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-glycyrrhetinic acid. Since this saponin has a 2'-O-β-D-glucopyranosyl moiety instead of the 2'-O-β-D-glucuronopyranosyl moiety of glycyrrhizin, the glucuronidation of 3-O-β-D-glucuronopyranosyl-glycyrrhetinic acid leading to glycyrrhizin is inhibited in this strain. All 4 offspring of the 83-555 strain produced glucoglycyrrhizin. Interestingly, 2 of the offspring produced both glycyrrhizin and glucoglycyrrhizin, and sequence analysis of the pkr gene suggested that these 2 offspring were hybrids of 83-555 strain and glycyrrhizin-producing strains.

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.

Matrix metalloproteinase-13 (MMP-13) is important in the pathology of osteoarthritis (OA). Although MMP-13 is considered a therapeutic target for OA, it is unclear how MMP-13 activity is regulated by the system that comprises various proteinases and their inhibitors. MMP-13 neutralizing antibodies could be a useful tool for investigating the involvement of MMP-13 in cartilage degeneration in OA-affected joints because antibodies possess high affinity and specificity compared with low-molecular weight chemical compounds. On the basis of three-dimensional structure and amino acid sequence information on MMP-13, we selected an appropriate antigen peptide and generated a neutralizing antibody by immunizing mice with the antigen. The most significant property of monoclonal antibody 14D10 was the specific binding to the active form of MMP-13, but not to the latent form, or other MMPs. With this property, active MMP-13 was measured selectively by an enzyme-linked immunosorbet assay. Furthermore, 14D10 suppressed the cleavage of type II collagen in human articular chondrocyte cultures, and 14D10 is thought to inhibit MMP-13 activity effectively. Thus, the highly selective MMP-13 neutralizing antibody (14D10) might be a useful tool for investigating the mechanism of type II collagen degradation in arthritic pathology.

Human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) have an endless self-renewal capacity and can theoretically differentiate into all types of lineages. They thus represent an unlimited source of cells for therapies of regenerative diseases, such as Duchenne muscular dystrophy (DMD), and for tissue repair in specific medical fields. However, at the moment, the low number of efficient specific lineage differentiation protocols compromises their use in regenerative medicine. We developed a two-step procedure to differentiate hESCs and dystrophic hiPSCs in myogenic cells. The first step was a culture in a myogenic medium and the second step an infection with an adenovirus expressing the myogenic master gene MyoD. Following infection, the cells expressed several myogenic markers and formed abundant multinucleated myotubes in vitro. When transplanted in the muscle of Rag/mdx mice, these cells participated in muscle regeneration by fusing very well with existing muscle fibers. Our findings provide an effective method that will permit to use hESCs or hiPSCs for preclinical studies in muscle repair.

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the ingestion of a variety of reef fish that have accumulated trace amounts of ciguatoxins produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50,000 people each year. The extremely low level of the causative neurotoxins, ciguatoxins, in fish has hampered the preparation of antibodies for detecting the toxins. In this paper, we describe a thiol strategy for synthesizing a keyhole limpet hemocyanin (KLH)-conjugate (20) of the ABCDE-ring fragment of the Pacific ciguatoxins, CTX1B (1) and 54-deoxyCTX1B (4). We succeeded in producing a monoclonal antibody (3G8) against the left wings of these ciguatoxins by immunizing mice with the hapten-KLH conjugate (20) as the synthetic antigen. The most promising mAb, 3G8, does not cross-react with other related marine toxins. Sandwich enzyme-linked immunosorbent assay (ELISA) utilizing 3G8 and the previously prepared monoclonal antibody (8H4) enabled us to detect 1 specifically at less than 0.28 ng/mL., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Recent evidences suggest that the extracts of plant products are able to modulate innate immune responses. A saponin GL and a chalcone ILG are representative components of Glycyrrhiza uralensis, which attenuate inflammatory responses mediated by TLRs. Here, we show that GL and ILG suppress different steps of the LPS sensor TLR4/MD-2 complex signaling at the receptor level. Extract of G. uralensis suppressed IL-6 and TNF-α production induced by lipid A moiety of LPS in RAW264.7 cells. Among various G. uralensis-related components of saponins and flavanones/chalcones, GL and ILG could suppress IL-6 production induced by lipid A in dose-dependent manners in RAW264.7 cells. Furthermore, elevation of plasma TNF-α in LPS-injected mice was attenuated by passive administration of GL or ILG. GL and ILG inhibited lipid A-induced NF-κB activation in Ba/F3 cells expressing TLR4/MD-2 and CD14 and BMMs. These components also inhibited activation of MAPKs, including JNK, p38, and ERK in BMMs. In addition, GL and ILG inhibited NF-κB activation and IL-6 production induced by paclitaxel, a nonbacterial TLR4 ligand. Interestingly, GL attenuated the formation of the LPS-TLR4/MD-2 complexes, resulting in inhibition of homodimerization of TLR4. Although ILG did not affect LPS binding to TLR4/MD-2, it could inhibit LPS-induced TLR4 homodimerization. These results imply that GL and ILG modulate the TLR4/MD-2 complex at the receptor level, leading to suppress LPS-induced activation of signaling cascades and cytokine production, but their effects are exerted at different steps of TLR4/MD-2 signaling.

Destroying aromaticity: A novel prenyltransferase (Trt2) involved in fungal meroterpenoid biosynthesis was shown to catalyze an unusual aromatic addition reaction onto a fully substituted aromatic ring. The prenylated product serves as a key intermediate in the biosynthesis of the most abundant series of meroterpenoids in fungi., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

SREBP-1 are ubiquitously expressed transcription factors, strongly expressed in lipogenic tissues where they regulate several metabolic processes like fatty acid synthesis. In skeletal muscle, SREBP-1 proteins regulate the expression of hundreds of genes, and we previously showed that their overexpression induced muscle atrophy together with a combined lack of expression of myogenic regulatory factors. Here we present evidences that SREBP-1 regulate muscle protein synthesis through the downregulation of the expression of MYOD1, MYOG and MEF2C factors. In myotubes overexpressing SREBP-1, restoring the expression of myogenic factors prevented atrophy and rescued protein synthesis, without affecting SREBP-1 action on atrogenes and proteolysis. Our results point out the roles of MRFs in the maintenance of the protein content and cell size in adult muscle fibre, and contribute to decipher the mechanisms by which SREBP-1 regulate muscle mass.

The aim of this study was to investigate the change of Integrin-linked kinase (ILK) expression of human retinal pigment epithelium (RPE) cells in response to high glucose, and the effect of targeting ILK with small interference RNA (siRNA) on the high glucose-induced expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1). The ILK mRNA and protein expression in human RPE cells were analyzed with RT-PCR and western blot after exposure to 5.5, 30, 40, 50 mM glucose, or 5.5 mM glucose+45.5 mM mannitol for 48 h. The expression of VEGF and ICAM-1 was also determined. Cells were treated with ILK siRNA, to determine the effect of ILK on VEGF and ICAM-1 expression following treatment with high glucose. High concentrations of glucose significantly up-regulated ILK mRNA and protein expression, and the ILK expression increased along with the glucose concentration. The changes of VEGF and ICAM-1 expression were similar to that of ILK expression. Knocking down ILK gene expression with siRNA inhibited the elevation of VEGF and ICAM-1 induced by high glucose treatment. These results suggested that ILK was involved in the response of RPE cells to high glucose and may therefore play a role in the pathogenesis of diabetic ophthalmology.

It is known that some kinase inhibitors are sensitive to the phosphorylation state of the kinase, and therefore those compounds can discriminate between a phosphorylated and unphosphorylated protein. In this study, we prepared two colony stimulating factor-1 receptor (CSF-1R) tyrosine kinase proteins: one highly phosphorylated by autophosphorylation and the other dephosphorylated by phosphatase treatment. These kinases were subjected to an activity-based assay to investigate the effect of their phosphorylation state on the potency of several kinase inhibitors. Dasatinib, sorafenib, PD173074 and staurosporine showed similar inhibition against different phosphorylation states of CSF-1R, but pazopanib, sunitinib, GW2580 and imatinib showed more potent inhibition against dephosphorylated CSF-1R. Binding analysis of the inhibitors to the two different phosphorylation forms of CSF-1R, using surface plasmon resonance spectrometry, revealed that staurosporine bound to both forms with similar affinity, but sunitinib bound to the dephosphorylated form with higher affinity. Thus, these observations suggest that sunitinib binds preferentially to the inactive form, preventing the activation of CSF-1R. Screening against different activation states of kinases should be an important approach for prioritizing compounds and should facilitate inhibitor design.

Expression of multiple genes involved in a particular metabolic pathway in a heterologous host facilitates the study of fungal secondary metabolite biosynthesis and production of useful compounds. Two plasmids with different selection markers, argB and the pyrithiamine resistance marker, are used to transform Aspergillus oryzae allowing for expression of up to three genes simultaneously.

Fragment-based lead discovery is a new approach for lead generation that has emerged in the past decade. Because the initial fragments identified in the fragment screening typically show weak binding affinity, an intensive medicinal chemistry effort would be required to grow initial fragments into a potential lead compound. Here we demonstrate a kinase focused evolved fragment (KFEF) library, constructed by click chemistry-based fragment assembly, that is a valuable source of kinase inhibitors. This combinatorial assembly of two fragments, kinase-privileged alkyne fragments and diversified azide fragments, by two cycloaddition reactions shows a unique potential for the one-step synthesis of structurally diverse evolved fragments. The screening of this triazole-based KFEF library allowed the rapid identification of potent lead candidates for FLT3 and GSK3β kinase., (Copyright © 2011 Elsevier Ltd. All rights reserved.)