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Spider dragline silk is a remarkably tough biomaterial and composed primarily of spidroins MaSp1 and MaSp2. During fiber self-assembly, the spidroin N-terminal domains (NTDs) undergo rapid dimerization in response to a pH gradient. However, obtaining a detailed understanding of this mechanism has been hampered by a lack of direct evidence regarding the protonation states of key ionic residues. Here, we elucidated the solution structures of MaSp1 and MaSp2 NTDs from Trichonephila clavipes and determined the experimental pKa values of conserved residues involved in dimerization using NMR. Surprisingly, we found that the Asp40 located on an acidic cluster protonates at an unusually high pH (∼6.5-7.1), suggesting the first step in the pH response. Then, protonation of Glu119 and Glu79 follows, with pKas above their intrinsic values, contributing toward stable dimer formation. We propose that exploiting the atypical pKa values is a strategy to achieve tight spatiotemporal control of spider silk self-assembly.

Introduction: Sensitive methods are necessary to identify the residual structure in an unfolded protein, which may be similar to the functionally native structure. Signal intensity in NMR experiments is useful for analyzing the line width for a dynamic structure; however, another contribution is contained. Methods: Here, the signal-intensity difference along the sequence was used for probability to calculate the standard deviation. Results: The relative values of the standard deviations were 0.57, 0.57, and 0.66 for alpha-synuclein wild-type, A53T, and A30P, respectively. This revealed that the flexible region was mainly in the Cterminal region of alpha-synuclein at higher temperatures as observed by the amide-proton exchange studies. Conclusion: In particular, the flexible structure was induced by the A30P mutation.

19F-NMR analysis using the optimized 19F chemical library enables the modeling of the structure of the weakly bound protein–compound complex, overcoming the difficulty in fragment-based drug discovery.

Summary Spatial isolation of molecules is often a powerful strategy for regulating their molecular behavior. Biological systems employ such mechanisms well; however, scientists have yet to rival nature, particularly for macromolecular substrates. We demonstrated that the encapsulation of a protein in a molecular cage with an open framework stabilizes the tertiary structure of the protein and improves its enzymatic activity. Particularly, when the three-dimensionally confined enzyme was exposed to an organic solvent, its half-life was prolonged 1,000-fold. Kinetic and spectroscopic analysis of the enzymatic reaction revealed that the key to this stability is the isolated space; this is reminiscent of chaperonins, which use their large internal cavities to assist the folding of client proteins. The single-molecule protein caging affords a new type of protein-based nanobiotechnology that accelerates molecular biology research as well as industrial applications.

Introduction Developing prognostic markers can be useful for clinical decision‐making. Peripheral blood (PB) examination is simple and basic that can be performed in any facility. We aimed to investigate whether PB examination can predict prognosis in coronavirus disease (COVID‐19). Methods Complete blood count (CBC) and PB cell morphology were examined in 38 healthy controls (HCs) and 40 patients with COVID‐19. Patients with COVID‐19, including 26 mild and 14 severe cases, were hospitalized in Juntendo University Hospital (Tokyo, Japan) between April 1 and August 6, 2020. PB examinations were performed using Sysmex XN‐3000 automated hematology analyzer and Sysmex DI‐60 employing the convolutional neural network‐based automatic image‐recognition system. Results Compared with mild cases, severe cases showed a significantly higher incidence of anemia, lymphopenia, and leukocytosis (P

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy—in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.

Fragment-based drug discovery (FBDD), which involves small compounds

Article, International journal of laboratory hematology. 43(2): 281-289(2021)

Spider dragline silk is well recognized due to its excellent mechanical properties. Dragline silk protein mainly consists of two proteins, namely, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). The MaSp N-terminal domain (NTD) conformation displays a strong dependence on ion and pH gradients, which is crucial for the self-assembly behavior of spider silk. In the spider major ampullate gland, where the pH is neutral and concentration of NaCl is high, the NTD forms a monomer. In contrast, within the spinning duct, where pH becomes more acidic (to pH ~ 5) and the concentration of salt is low, NTD forms a dimer in antiparallel orientation. In this study, we report near-complete backbone and side chain chemical shift assignment of the monomeric form of NTD of MaSp2 from Nephila clavipes at pH 7 in the presence of 300 mM NaCl. Our NMR data demonstrate that secondary structure of monomeric form of NTD MaSp2 consists of five helix regions.

HIV-1 capsid is comprised of over a hundred p24 protein molecules, arranged as either pentamers or hexamers. Three p24 mutants with amino acid substitutions in capsid N-terminal domain protein were examined: G60W (α3-4 loop), M68T (helix 4), and P90T (α4-5 loop), which exhibited no viability for biological activity. One common structural feature of the three p24 N-domain mutants, examined by NMR, was the long-range effect of more β-structures at the β2-strand in the N-terminal region compared with the wild-type. In addition, the presence of fewer helical structures was observed in M68T and P90T, beyond the broad area from helix 1 to the C-terminal part of helix 4. This suggests that both N-terminal beta structures and helices play important roles in the formation of p24 hexamers and pentamers. Next, compared with P90T, we examined cis-conformation or trans-conformation of wild-type adopted by isomerization at G89-P90. Since P90T mutant adopts only a trans-conformation, comparison of chemical shifts and signal intensities between each spectra revealed that the major peaks (about 85%) in the spectrum of wild-type correspond to trans-conformation. Furthermore, it was indicated that the region in cis-conformation (minor; 15%) was more stabilized than that observed in trans-conformation, based on the analyses of heteronuclear Overhauser effect as well as the order-parameter. Therefore, it was concluded that the cis-conformation is more favorable than the trans-conformation for the interaction between the p24 N-terminal domain and cyclophilin-A. This is because HIV-1 with a P90T protein, which adopts only a trans-conformation, is associated with non-viability of biological activity.

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis reveals that SptF catalyzes four continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced novel cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF reveal the molecular basis of the enzyme reactions, and suggest that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

The effect of ions on the structure and dynamics of a spider silk protein is elucidated. Chaotropic ions prevent intra- and inter-molecular interactions on the repetitive domain, which are required to maintain the solubility, while kosmotropic ions promote hydrogen bond interactions in the glycine-rich region, which are a prerequisite for β-sheet formation.

Polyampholytes are emerging macromolecular membrane non-penetrating cryoprotectants; however, the mechanism behind their cryopreservation remains unclear. Here, we investigated the mechanism using solid-state NMR spectroscopy. The polymer-chain dynamics and the water and ion mobilities in the presence of various membrane penetrating and non-penetrating cryoprotectants were monitored at low temperatures to mimic cryopreservation conditions. NMR experiments revealed that the water, Sodium-ion, and polymer-chain signals in a carboxylated poly-ʟ-lysine (COOH-PLL) solution broadened upon cooling, indicating increasingly restricted mobility and increased solution viscosity. Moreover, strong intermolecular interactions facilitated the COOH-PLL glass transition, trapping water and salt in the gaps of the reversible matrix, preventing intracellular ice formation and osmotic shock during freezing; this reduced cell stress is responsible for cryoprotection. This simple NMR technique enabled the correlation of the cryoprotective properties of polymers that operate through mechanisms different from those of current cryoprotectants, and will facilitate the future molecular design of cryoprotectants. Cellular cryopreservation is an important tool in modern biology. Here, previously reported polyampholyte cryoprotectants are studied by solid-state NMR, revealing the molecular mechanisms at play.

Two different types of polycyclic ether toxins, namely brevisulcenals (KBTs) and brevisulcatic acids (BSXs), produced by the red tide dinoflagellate Karenia brevisulcata, were the cause of a toxic incident that occurred in New Zealand in 1998. Four major components, KBT-F, -G, -H, and -I, shown to be cytotoxic and lethal in mice, were isolated from cultured K. brevisulcata cells, and their structures were elucidated by spectroscopic analyses. New analogues, brevisulcenal-A1 (KBT-A1) and brevisulcenal-A2 (KBT-A2), toxins of higher polarity than that of known KBTs, were isolated from neutral lipophilic extracts of bulk dinoflagellate culture extracts. The structures of KBT-A1 and KBT-A2 were elucidated as sulfated analogues of KBT-F and KBT-G, respectively, by NMR and matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI TOF/TOF), and by comparison with the spectra of KBT-F and KBT-G. The cytotoxicities of the sulfate analogues were lower than those of KBT-F and KBT-G.

Two different types of polycyclic ether toxins, namely brevisulcenals (KBTs) and brevisulcatic acids (BSXs), produced by the red tide dinoflagellate Karenia brevisulcata, were the cause of a toxic incident that occurred in New Zealand in 1998. Four major components, KBT-F, -G, -H, and -I, shown to be cytotoxic and lethal in mice, were isolated from cultured K. brevisulcata cells, and their structures were elucidated by spectroscopic analyses. New analogues, brevisulcenal-A1 (KBT-A1) and brevisulcenal-A2 (KBT-A2), toxins of higher polarity than that of known KBTs, were isolated from neutral lipophilic extracts of bulk dinoflagellate culture extracts. The structures of KBT-A1 and KBT-A2 were elucidated as sulfated analogues of KBT-F and KBT-G, respectively, by NMR and matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI TOF/TOF), and by comparison with the spectra of KBT-F and KBT-G. The cytotoxicities of the sulfate analogues were lower than those of KBT-F and KBT-G.

This study investigated the interaction of borate ions released from surface pre-reacted glass ionomer (S-PRG) fillers with the biological apatites of enamel and dentin using solid-state (SS) magic-angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy analysis. We further evaluated the effect of borate ions on the pH change. Bovine enamel and dentin powder were submerged in S-PRG eluate (with borate ion concentration of 100 mM) for 3 h, then washed with deionized water (DW). The dried enamel and dentin specimens were used for 11B SS-NMR and Fourier transform infrared spectroscopy (FT-IR) analysis. Enamel and dentin blocks were treated with three solutions: DW (control), S-PRG eluent and borate solution (100 mM). The treated samples were submerged in the demineralization solution and the pH was measured using a pH meter daily for 6 days. The surfaces of the enamel and dentin blocks were then observed using field emission scanning electron microscopy (FE-SEM). SS-NMR analysis revealed the presence of adsorbed borate ions in the enamel and dentin samples in a tetra-coordinated form. The pH results demonstrated an increase in pH values in the S-PRG and borate groups. SEM images showed that the surfaces of the control group were demineralized, whereas the surfaces of the S-PRG and borate groups were intact. These results concluded that borate ions could be adsorbed to enamel and dentin in the tetra-coordinated form. Borate ions possess a buffer capacity which may help to protect the tooth structure against acid attacks and promote remineralization.

Lanthanide ions (Ln3+) show similar physicochemical properties in aqueous solutions, wherein they exist as + 3 cations and exhibit ionic radii differences of less than 0.26 Å. A flexible linear peptide lanthanide binding tag (LBT), which recognizes a series of 15 Ln3+, shows an interesting characteristic in binding specificity, i.e., binding affinity biphasically changes with an increase in the atomic number, and shows a greater than 60-fold affinity difference between the highest and lowest values. Herein, by combining experimental and computational investigations, we gain deep insight into the reaction mechanism underlying the specificity of LBT3, an LBT mutant, toward Ln3+. Our results clearly show that LBT3-Ln3+ binding can be divided into three, and the large affinity difference is based on the ability of Ln3+ in a complex to be directly coordinated with a water molecule. When the LBT3 recognizes a Ln3+ with a larger ionic radius (La3+ to Sm3+), a water molecule can interact with Ln3+ directly. This extra water molecule infiltrates the complex and induces dissociation of the Asn5 sidechain (one of the coordinates) from Ln3+, resulting in a destabilizing complex and low affinity. Conversely, with recognition of smaller Ln3+ (Sm3+ to Yb3+), the LBT3 completely surrounds the ions and constructs a stable high affinity complex. Moreover, when the LBT3 recognizes the smallest Ln3+, namely Lu3+, although it completely surrounds Lu3+, an entropically unfavorable phenomenon specifically occurs, resulting in lower affinity than that of Yb3+. Our findings will be useful for the design of molecules that enable the distinction of sub-angstrom size differences.

A new unique sesquiterpene lactone, bicyclolamellolactone A (1), was isolated together with two known monocyclofarnesol-type sesquiterpenes, lamellolactones A (2) and B (3), from the Indonesian marine sponge Lamellodysidea sp. (cf. L. herbacea). The planar structure of 1 was assigned based on its spectroscopic data (1D and 2D NMR, HRESIMS, UV, and IR spectra). The relative and absolute configuration of 1 was determined by comparison of its calculated and experimental electronic circular dichroism spectra in combination with NOESY correlations. Compounds 1–3 inhibited bone morphogenic protein (BMP)-induced alkaline phosphatase activity in mutant BMP receptor-carrying C2C12 cells with IC50 values of 51, 4.6, and 20 μM, respectively.

Spider dragline silk is well recognized due to its excellent mechanical properties. Dragline silk protein mainly consists of two proteins, namely, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). The MaSp N-terminal domain (NTD) conformation displays a strong dependence on ion and pH gradients, which is crucial for the self-assembly behavior of spider silk. In the spider major ampullate gland, where the pH is neutral and concentration of NaCl is high, the NTD forms a monomer. In contrast, within the spinning duct, where pH becomes more acidic (to pH ~ 5) and the concentration of salt is low, NTD forms a dimer in antiparallel orientation. In this study, we report near-complete backbone and side chain chemical shift assignment of the monomeric form of NTD of MaSp2 from Nephila clavipes at pH 7 in the presence of 300 mM NaCl. Our NMR data demonstrate that secondary structure of monomeric form of NTD MaSp2 consists of five helix regions.

The residual solution structures of two alpha-synuclein mutants, A30P and A53T, observed in family members of patients with Parkinson's disease were compared with that of wild-type by NMR. The A53T substitution had been shown to accelerate fibril formation of alpha-synuclein, whereas the A30P mutation has the negative and positive effects on the formation of the fibril and spherical oligomer, respectively. The remaining structure was analyzed via amide-proton exchange and signal intensity measurements using NMR. Amide-proton exchange was used for both the calculation of kex values and ratio of kex at different temperatures. Effects of the A30P (N-terminal region) mutation were observed at the C-terminal region as a more flexible structure, suggesting that long-range interactions exist between the N- and C-terminal regions in alpha-synuclein. In addition, the N-terminal region adopted a more rigid structure in the A53T and A30P mutants than in the wild-type. It was concluded that the structural change caused by the mutations is related to the formation of a beta-hairpin at the initiation site of the N-terminal core structure. Furthermore, the signal intensity was used to estimate the rigidity of the structure. Higher signal intensities were observed for A30P at the 112, 113, and 116 C-terminal residues, suggesting that this region adopts more flexible structure. The ratio of the intensities at different temperatures indicated more flexible or rigid structures in the N-terminal region of A30P than in that of wild-type. Thus, using different approaches and temperatures is a good method to analyze residual structure in intrinsically disordered proteins.

Mycolic acid-containing bacteria (MACB) are known to activate cryptic natural product biosynthesis in co-cultures with actinobacteria. We cultured Actinosynnema mirum NBRC 14064, a producer of the mono-cyclic polyene macrolactam mirilactam A (6), with the MACB Tsukamurella pulmonis TP-B0596. As a result, three novel compounds (mirilactams C-E, 1-3) were produced in the co-culture conditions. Compounds 1-3 were likely derived from 6 by epoxidation and subsequent spontaneous cyclization. The chemical structures and stereochemistries of 1-3 were determined by spectroscopic analyses (NMR and MS), conformational searches in the optimized potentials for liquid simulations-3 (OPLS3) force field, and calculations of electronic circular dichroism (ECD).

Marine organisms such as marine sponges and soft corals are valuable sources of pharmacologically active secondary metabolites. In our ongoing research on the discovery of new secondary metabolites from marine organisms, two new pyrrolo-2-aminoimidazoles, clathriroles A (1) and B (2), were isolated from the water-soluble portion prepared from the methanol and acetone (2:1) extract of the marine sponge, Clathria prolifera, collected in Myanmar. The chemical structures of the isolated compounds were determined using extensive spectroscopic techniques, including NMR, HRESIMS, IR, and optical rotation, and comparisons with the reported literature. The spectroscopic analyses of 1 and 2 suggested that 1 is an enantiomer of antifungal N-methylmanzacidin C isolated from the marine sponge Axinella brevistyla, whereas 2 is a diastereomer of manzacidin D at C-11 isolated from the marine sponge Astrosclera willeyana. To the best of our knowledge, this is the first report of the isolation of the pyrrolo-2-aminoimidazole compounds from C. prolifera. Furthermore, in contrast to the potency of N-methylmanzacidin C against Saccharomyces cerevisiae, the antifungal assay revealed that 1 and 2 lack any activity against this strain. Thus, these observations may suggest that the absolute configurations at both C-9 and C-11 play an important role in controlling the antifungal activity of this type of compound.

New polycyclic tetramate macrolactams, Umezawamides A (1) and B (2) were isolated from a combined-culture of Umezawaea sp. RD066910 and mycolic-acid containing bacterium Tsukamurella pulmonis TP-B0596. Their planar structures and partial stereochemistries were determined based on the spectroscopic analysis, MMFF conformational search, and ECD calculations. Umezawamides are the first secondary metabolites isolated from the genus Umezawaea and they exhibited cytotoxicities to P388 murine leukemia cells. Furthermore, umezawamide A (1) showed growth inhibitory activity against Candida albicans.

Nocardia is a potent bacterial producer of bioactive compounds. From a culture of Nocardia beijingensis NBRC 16342, we isolated four aromatic compounds, named beijinchromes A-D (1-4). We purified them by silica gel chromatography and reverse phase HPLC, and identified their structures by NMR and high resolution (HR)-MS analyses. 1, 2, and 4 are novel 1,2,3,8-tetrasubstituted naphthalenes, and 3 is a novel 3,8-disubstituted ortho-naphthoquinone. 1 and 2 exert antioxidant activities, and 3 exhibits antibiotic activity. Remarkably, the putative biosynthetic gene clusters for 1-4 are widely distributed in 37 Nocardia species, implying their potential to produce this family of compounds and important biological functions of beijinchromes.

Amphidinols are polyketide metabolites produced by marine dinoflagellates and are chiefly composed of a long linear chain with polyol groups and polyolefins. Two new homologues, amphidinols 20 (AM20, 1) and 21 (AM21, 2), were isolated from Amphidinium carterae collected in Korea. Their structures were elucidated by detailed NMR analyses as amphidinol 6-type compounds with remarkably long polyol chains. Amphidinol 21 (2) has the longest linear structure among the amphidinol homologues reported so far. The congeners, particularly amphidinol 21 (2), showed weaker activity in hemolysis and antifungal assays compared to known amphidinols.

This paper uses a two-period overlapping generations model in order to provide a theoretical design for the optimal public pension system based on a partial equilibrium analysis. Household preferences depend on two periods' consumption and leisure and are positively homogeneous of degree m with respect to consumption in the working and retired periods. We present characteristic features of the optimal public pension system in this paper. First, differences in the population growth rate do not affect the large/small relation between the optimal net lifetime burden rates of generations. Second, we present the optimal public pension system explicitly if m

Nocardia is a potent bacterial producer of bioactive compounds. From a culture of Nocardia beijingensis NBRC 16342, we isolated four aromatic compounds, named beijinchromes A-D (1-4). We purified them by silica gel chromatography and reverse phase HPLC, and identified their structures by NMR and high resolution (HR)-MS analyses. 1, 2, and 4 are novel 1,2,3,8-tetrasubstituted naphthalenes, and 3 is a novel 3,8-disubstituted ortho-naphthoquinone. 1 and 2 exert antioxidant activities, and 3 exhibits antibiotic activity. Remarkably, the putative biosynthetic gene clusters for 1-4 are widely distributed in 37 Nocardia species, implying their potential to produce this family of compounds and important biological functions of beijinchromes.

Three new lignoids, premnan A (1), premnan B (2), and tauntangyiol C (3), were isolated from Premna serratifolia wood, a traditional cosmetic plant in Myanmar, together with a new lignoid, premnan C (4) assumed to be an artifact, one natural new lignoid (5), and three known lignoids (6–8). The structures of the new compounds 1–4 were elucidated based on 1D and 2D NMR, IR spectroscopy, and HRESIMS. The absolute configurations of 1–4 were also determined by optical rotation, circular dichroism (CD) data analyses, and comparisons with the reported literature. All isolated compounds were tested for their melanogenesis activities against the B16-F10 mouse melanoma cell line. Compounds 1 and 4 showed melanogenesis enhancing activities of 31% and 50%, respectively, at a 50 μM concentration. Compounds 2, 3, and 6 increased melanin production by 67%, 30%, and 45%, respectively, at a 100 μM concentration, without any cytotoxicity.

Sesterterpenoids are a group of terpenoid natural products that are primarily biosynthesized via cyclization of the C25 linear substrate geranylfarnesyl pyrophosphate (GFPP). Although the long carbon chain of GFPP in theory allows for many different cyclization patterns, sesterterpenoids are relatively rare species among terpenoids, suggesting that many intriguing sesterterpenoid scaffolds have been overlooked. Meanwhile, the recent identification of the first sesterterpene synthase has allowed the discovery of new sesterterpenoids by the genome mining approach. In this study, we characterized the unusual fungal sesterterpene synthase EvQS and successfully obtained the sesterterpene quiannulatene (1) with a novel and unique highly congested carbon skeleton, which is further oxidized to quiannulatic acid (2) by the cytochrome P450 Qnn-P450. A mechanistic study of its cyclization from GFPP indicated that the biosynthesis employs an unprecedented cyclization mode, which involves three rounds of hydride shifts and two successive C-C bond migrations to construct the 5-6-5-5-5 fused ring system of 1.

Cryopreservation enables long-term preservation of cells at ultralow temperatures. Current cryoprotective agents (CPAs) have several limitations, making it imperative to develop CPAs with advanced properties. Previously, we developed a novel synthetic polyampholyte-based CPA, copolymer of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and methacrylic acid(MAA) (poly(MAA-DMAEMA)), which showed excellent efficiency and biocompatibility. Introduction of hydrophobicity increased its efficiency significantly. Herein, we investigated the activity of other polyampholytes. We prepared two zwitterionic polymers, poly(sulfobetaine) (SPB) and poly(carboxymethyl betaine) (CMB), and compared their efficiency with poly(MAA-DMAEMA). Poly-SPB showed only intermediate property and poly-CMB showed no cryoprotective property. These data suggested that the polymer structure strongly influences cryoprotection, providing an impetus to elucidate the molecular mechanism of cryopreservation. We investigated the mechanism by studying the interaction of polymers with cell membrane, which allowed us to identify the interactions responsible for imparting different properties. Results unambiguously demonstrated that polyampholytes cryopreserve cells by strongly interacting with cell membrane, with hydrophobicity increasing the affinity for membrane interaction, which enables it to protect the membrane from various freezing-induced damages. Additionally, cryoprotective polymers, especially their hydrophobic derivatives, inhibit the recrystallization of ice, thus averting cell death. Hence, our results provide an important insight into the complex mechanism of cryopreservation, which might facilitate the rational design of polymeric CPAs with improved efficiency.

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene (1), having a 6-8-6-5-fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope-incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation-initiated second cyclization sequence, 1,2-alkyl migration, and 1,5-hydride shift to generate the novel scaffold of 1.

Although periodate-oxidized dextran is widely used in biomedical applications, the degradation mechanism of oxidized dextran has not yet been elucidated. Herein, we propose a novel main chain scission mechanism of oxidized dextran triggered by reaction with amine. NMR analysis revealed four hemiacetal substructures during oxidation by periodate. Kinetic analysis showed that the degradation time constant of the C3-removed substructure and increasing time constant of the reducing end protons are consistent with the decrease in molecular weight determined by gel permeation chromatography. A methylene group is generated at the same time constant of degradation, indicating that oxidized dextran degradation proceeds via a Maillard reaction. Oxidized dextran does not degrade in saline solution without reactive amine species. Thus, we conclude that oxidized dextran is degraded in the main chain via Schiff base formation through a Maillard reaction, depending on the oxidation ratio and amino acid concentration. These findings help to elucidate the reaction mechanism of polysaccharide degradation and develop novel biodegradable polysaccharide materials for biomedical applications. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 2254–2260

A terrestrial bacterium, Streptomyces sp. NZ-6, produced niizalactams A-C (1-3), unprecedented di- and tricyclic macrolactams, by coculturing with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. Their complete structures, including absolute configurations, were elucidated on the basis of spectroscopic data and chemical derivatization. Their unique skeletons are proposed to be biosynthesized from a common 26-membered macrolactam intermediate by SN2 cyclization or an intramolecular Diels-Alder reaction.

Using activity guided purification, four known compounds, sesquiterpene atractylenolide III (1), and the polyacetylenes 14-acetoxy-12-senecioyloxytetradeca-2E,8E,10E-trien-4,6-diyn-1-ol (2), 14-acetoxy-12-α-methylbutyl-2E,8E,10E-trien-4,6-diyn-1-ol (3), and 14-acetoxy-12-β -methylbutyl-2E,8E,10E-trien-4,6-diyn-1-ol (4), were isolated from a traditional herbal medicine, Atractylodes rhizome. Structurally similar 3 and 4 (3/4 mixture) were obtained as a mixture. In intact Chinese hamster ovary (CHO) K1 cell assays, 1, 2, and a 3/4 mixture selectively inhibited cholesterol [14C]oleate synthesis from [14C]oleate with IC50 values of 73.5 µM, 35.4 µM, and 10.2 µM, respectively, without any effects on cytotoxicity. As a potential target of these inhibitors involved in cholesteryl ester (CE) synthesis, effects on sterol O-acyltransferase (SOAT) activity were investigated using microsomes prepared from CHO-K1 cells as an enzyme source. Hence, these compounds inhibit SOAT activity with IC50 values (211 µM for 1, 29.0 µM for 2, and 11.8 µM for 3/4 mixture) that correlate well with those measured from intact cell assays. Our results strongly suggest that these compounds inhibit CE synthesis by blocking SOAT activity in CHO-K1 cells.

The objective of this study is to control and elucidate the mechanism of molecular degradation in a polysaccharide hydrogel. Glycidyl methacrylate (GMA) immobilized dextran (Dex-GMA) was oxidized by periodate to introduce aldehyde groups (oxidized Dex-GMA). The hydrogel was formed by the addition of dithiothreitol to the oxidized Dex-GMA solution through thiol Michael addition with the preservation of the aldehyde group for degradation points. It was experimentally determined that the degradation of this hydrogel can be controlled by the addition of amino groups and the speed of degradation can be controlled independently of mechanical properties because crosslinking and degradation points are different. In addition, the molecular mechanism of the crosslinking between the thiol and aldehyde groups was found to control the degradation of dextran derivatives. It is expected that these results will be beneficial in the design of polymer materials in which the speed of degradation can be precisely controlled. In addition, the cytotoxicity of oxidized Dex-GMA was approximately 3000 times lower than that of glutaraldehyde. The low cytotoxicity of the aldehyde in oxidized Dex-GMA was the likely reason for the harmless functionalized polysaccharide material. Possible future clinical applications include cell scaffolds in regenerative medicine and carriers for drug delivery systems.

Mycolic acid-containing bacteria (MACB) are known to activate cryptic natural product biosynthesis in co-cultures with actinobacteria. We cultured Actinosynnema mirum NBRC 14064, a producer of the mono-cyclic polyene macrolactam mirilactam A (6), with the MACB Tsukamurella pulmonis TP-B0596. As a result, three novel compounds (mirilactams C-E, 1-3) were produced in the co-culture conditions. Compounds 1-3 were likely derived from 6 by epoxidation and subsequent spontaneous cyclization. The chemical structures and stereochemistries of 1-3 were determined by spectroscopic analyses (NMR and MS), conformational searches in the optimized potentials for liquid simulations-3 (OPLS3) force field, and calculations of electronic circular dichroism (ECD).

The β-sheet is the key structure underlying the excellent mechanical properties of spider silk. However, the comprehensive mechanism underlying β-sheet formation from soluble silk proteins during the transition into insoluble stable fibers has not been elucidated. Notably, the assembly of repetitive domains that dominate the length of the protein chains and structural features within the spun fibers has not been clarified. Here we determine the conformation and dynamics of the soluble precursor of the repetitive domain of spider silk using solution-state NMR, far-UV circular dichroism and vibrational circular dichroism. The soluble repetitive domain contains two major populations: ~65% random coil and ~24% polyproline type II helix (PPII helix). The PPII helix conformation in the glycine-rich region is proposed as a soluble prefibrillar region that subsequently undergoes intramolecular interactions. These findings unravel the mechanism underlying the initial step of β-sheet formation, which is an extremely rapid process during spider silk assembly., β-sheet structure underlies the mechanical properties of spider silk but the mechanism to form β-sheet from soluble silk protein during transition into insoluble fibers has not been elucidated. Here the authors unravel the mechanism of β-sheet formation using NMR and circular dichroism spectroscopy.

Four new catechol derivatives, hinduchelins A–D (1–4), composed of 2,3- dihydroxybenzoic acid, threonine, and decarboxylated phenylalanine, were isolated from Streptoalloteichus hindustanus. Their structures and absolute configurations were elucidated by interpretation of NMR and HRMS data and quantum chemical ECD calculations. The iron-binding properties of the compounds were evaluated by a pyoverdine production assay in Pseudomonas aeruginosa, and compound 4 showed moderate ability to induce pyoverdine production at 50 μM. None of the compounds were cytotoxic toward HL-20, A549, SMMC-7721, MCF-7, and SW-480 tumor cell lines.