An Isocytidine Derivative with a 2‐Amino‐6‐methylpyridine Unit for Selective Recognition of the CG Interrupting Site in an Antiparallel Triplex DNA

Sequence‐specific recognition of duplex DNA mediated by triple helix formation offers a potential basis for oligonucleotide therapy and biotechnology. However, triplex formation is limited mostly to homopurine strands, due to poor stabilization at CG or TA base pairs in the target duplex DNA sequences. Several non‐natural nucleosides have been designed for the recognition of CG or TA base pairs within an antiparallel triplex DNA. Nevertheless, problems including low selectivity and high dependence on the neighboring bases remain unsolved. We thus synthesized N²‐arylmethyl isodC derivatives and incorporated them into triplex‐forming oligonucleotides (TFOs) for the selective recognition of the CG base pair within antiparallel triplex DNA. It was shown that an isodC derivative bearing a 2‐amino‐6‐methylpyridine moiety (AP‐isodC) recognizes the CG base pair with high selectivity in antiparallel triplex DNA irrespective of the flanking base pairs.     

Efficient DNA strand displacement by a W-shaped nucleoside analogue (WNA-βT) containing an ortho-methyl-substituted phenyl ring

Molecules that can target duplex DNA with sequence selectivity have the potential to be useful tools in genomic research and also as therapeutic agents. Homopurine-homopyrimidine stretches in duplex DNA can be recognized by homopurine or homopyrimidine TFOs (triplex-forming oligonucleotides) through the formation of triplex DNA. We have previously developed bicyclic nucleoside analogues (WNAs) for the formation of stable triplexes in the formation of stable antiparallel triplexes containing a TA or a CG interrupting site. In this study, we investigated the effects on triplex DNA formation of ortho-, meta-, and para-methyl substituent groups on the aromatic ring of the WNA analogue. It was found that the homopurine TFO containing meta- and para-methyl-substituted WNA-βT (mMe-WNA-βT, pMe-WNA-βT) stabilized triplexes containing a TA interrupting site or a GC site, respectively. Interestingly, the ortho-methyl-substituted WNA-βT (oMe-WNA-βT) efficiently promoted DNA strand displacement to form the TFO/pyrimidine duplex. A detailed investigation showed that the duplex was in the antiparallel orientation and that its formation took place prior to triplex formation with the need for a magnesium cation. NOESY measurements indicated a significant difference in the rotation flexibilities of the phenyl rings of WNA-βTs: that is, the conformation of the ortho-methylated phenyl ring was stable in a temperature-independent manner. It was speculated that the initial formation of a ternary complex was followed by strand displacement and then the formation of the TFO/pyrimidine duplex together with the TFO(2)/pyrimidine triplex formation during the early stage, and that the equilibrium shifted to the triplex during the later stage. Although the detailed role is still uncertain, the fixed phenyl ring of oMe-WNA-βT might play a role in the displacement reaction.     

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis

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Seasonal change in the level and the chemical forms of aluminum in soil solution under a Japanese cedar forest

The level of dissolved aluminum and its chemical forms in soil solutions consecutively collected by a porous cup vacuum sampler were monitored over a period from January 2001 to December 2001 at a Japanese cedar (Cryptomeria japonica) forestry area susceptible to acid deposition to characterize current soil dynamics and to evaluate potential tree damages. Distinction and characterization of Al species with differential toxicities were performed by two complementary speciation techniques; cation-exchange HPLC with fluorometric detection using 8-hydroxyquinoline-5-sulfonic acid (HQS) and size-fractionation/inductively coupled plasma atomic emission spectrometry (ICP-AES). The concentrations of free Al (mainly Al3+ and Al(OH)2+) and inert Al (existing as the complexed and/or colloidal forms) ranged between 0-150 microM and 10-50 microM, respectively. The concentrations of inert Al were mostly below 40 microM during an annual cycle and showed no marked seasonal variation, while free Al concentrations showed a clear tendency to increase in the spring and summer seasons (in the period from April to August) probably due to the enhanced activity of microbial nitrification and the resultant soil acidification. Major cations and anions were also regularly determined and their seasonal changes were correlated with that of the dissolved Al concentration. Correlations between total Al (mainly existing as free Al) and the related species (and environmental conditions) were as follows: Al and Mg (R=0.96, P<0.01), Al and Ca (R=0.97, P<0.01), Al and NO3- (R=0.68, P<0.01), Al and temperature (R=0.68, P<0.01), Al and solution pH (R=-0.61, P<0.01), solution pH and NO3- (R=-0.65, P<0.01).     

Identification of fungi using DNA sequences: an approach to identify Fusarium species isolated from domestic unpolished rice

Molecular approaches are being developed to provide for the rapid and objective identification of fungi. We attempted the identification of Fusarium species by a genetic analysis to validate practically the utility of a molecular approach for fungal identification and to reveal its limitations, and sequenced three regions, the 5' end of the 28S rRNA gene (D2 region) and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) regions, in the rRNA genes. The DNA sequences of 38 Fusarium strains isolated from domestic unpolished rice were compared for similarity with entries in the GenBank. Based on this comparison, it was estimated that all these three regions, as a minimum, must be compared with the database to identify Fusaria at the species level. According to the combinations of sequences in the three regions, the 38 isolates were classified into 13 groups. Out of the 13 groups, 6 groups (20 isolates in total) could be identified as definite species based only on the sequence data. For the other 6 groups (17 isolates in total), candidate species were limited on the basis of the sequence similarity, and then the isolates were identified at the species level with the aid of morphology. Only one isolate could not be identified. These results verified that DNA sequence comparison with the GenBank database is useful for the identification of Fusarium species.     

Antioxidant flavonol glycosides in mulberry (Morus alba L.) leaves isolated based on LDL antioxidant activity

Oxidation of low-density lipoprotein (LDL) has been implicated in atherogenesis. Antioxidants that prevent LDL from oxidation may reduce atherosclerosis. We investigated LDL antioxidant activity and extracted compounds of mulberry (Morus alba L.) leaves. The LDL antioxidant activity of 60% ethanol extracted of mulberry leaves, which inhibits human LDL oxidation induced by copper ion, was determined on the basis of oxidation lag time and calculated as epigallocatechin 3-gallate equivalents (58.3 μmol of EGCG equivalent/g of dry weight). Three flavonol glycosides [quercetin 3-(6-malonylglucoside), rutin (quercetin 3-rutinoside) and isoquercitrin (quercetin 3-glucoside)] were identified as the major LDL antioxidant compounds by LC-MS and NMR. The amounts of these flavonol glycosides in mulberry leaves and mulberry-leaf tea were determined by HPLC. Our results showed that quercetin 3-(6-malonylglucoside) and rutin were the predominant flavonol glycosides in the mulberry leaves.     

MORI-A CPS: 3D printed soft actuators with 4D assembly simulation

Artificial Life and Robotics - Soft modular robotics combines soft materials and modular mechanisms. We are developing a vacuum-driven actuator module, MORI-A, which combines a 3D-printed flexible...     

Chemoproteomic Profiling Reveals that Anticancer Natural Product Dankastatin B Covalently Targets Mitochondrial VDAC3**

Chlorinated gymnastatin and dankastatin alkaloids derived from the fungal strain Gymnascella dankaliensis have been reported to possess significant anticancer activity but their mode of action is unknown. These members possess electrophilic functional groups that can might undergo covalent bond formation with specific proteins to exert their biological activity. To better understand the mechanism of action of this class of natural products, we mapped the proteome-wide cysteine reactivity of the most potent of these alkaloids, dankastatin B, by using activity-based protein profiling chemoproteomic approaches. We identified a primary target of dankastatin B in breast cancer cells as cysteine C65 of the voltage-dependent anion-selective channel on the outer mitochondrial membrane VDAC3. We demonstrated direct and covalent interaction of dankastatin B with VDAC3. VDAC3 knockdown conferred hypersensitivity to dankastatin B-mediated antiproliferative effects in breast cancer cells, thus indicating that VDAC3 was at least partially involved in the anticancer effects of this natural product. Our study reveals a potential mode of action of dankastatin B through covalent targeting of VDAC3 and highlights the utility of chemoproteomic approaches in gaining mechanistic understanding of electrophilic natural products.     

3D-Printed Methane-Producing Electrodes for Microbial Fuel Cells Developed Using Biogel Ink Containing Live Methanogens and White Charcoal

Methanogens are used as catalysts for cathodes in microbial fuel cells, to reduce CO₂ to CH₄. However, the attachment of microbes to the electrodes via culturing is time-consuming, and inadequate biofilm formation can lead to lesser surface area coverage, resulting in reduced methane formation. This study aims to improve the production efficiency and performance of methanogen cathodes developed using 3D printing of bioink containing live methanogens. A progressive cavity pump is used for the 3D gel-printing of methanogens and micro-sized white charcoal particles into the desired structure. Despite the absence of anaerobic conditions during printing, the 3D-printed cathodes with higher concentrations of microbial inoculum in the bioink produce more methane gas. Even with an unconcentrated inoculum, the methanogens multiply 800-fold during incubation, resulting in increased methane gas production. The predominant methanogens in the electrodes included the hydrogenotrophic Methanobacterium spp. Therefore, the technique used in this study can be used to successfully develop 3D-printed biocathodes catalyzed by methanogenic microbes with verifiable practical applicability. This study is the first to report the growth of methanogens and their methanogenic activity in 3D-printed cathodes.