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971.
972.
973.
Takuya Koseki Keiji Mochizuki Hiroe Kisara Akimasa Miyanaga Shinya Fushinobu Tetsuya Murayama Yoshihito Shiono 《Applied microbiology and biotechnology》2010,86(1):155-161
We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 α-l-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (M
r) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose
side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant
difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release
of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA
alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion,
the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring
about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application
in the degradation of polysaccharides. 相似文献
974.
Noriko Sumi Tsuyoshi Nishioku Fuyuko Takata Junichi Matsumoto Takuya Watanabe Hideki Shuto Atsushi Yamauchi Shinya Dohgu Yasufumi Kataoka 《Cellular and molecular neurobiology》2010,30(2):247-253
The blood–brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons.
BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with
microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide
(LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia.
When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the
RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased
in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like
immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These
functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction
of the BBB by producing reactive oxygen species through NADPH oxidase. 相似文献
975.
Hiroshi Takeshita Jun Watanabe Yoichi Kimura Katsuhiro Kawakami Hisashi Takahashi Makoto Takemura Akihiro Kitamura Kazuhiko Someya Ryohei Nakajima 《Bioorganic & medicinal chemistry letters》2010,20(13):3893-3896
Based on the HTS hit compound 1a, an inhibitor of β-1,6-glucan synthesis, we synthesized novel pyridobenzimidazole derivatives and evaluated their antifungal activity. Among the compounds synthesized, we identified the potent compound 15e, which exhibits excellent activity superior to fluconazole against both Candida glabrata and Candida krusei. From the SAR study, we revealed essential moieties for antifungal activity. 相似文献
976.
977.
Satoshi Kitamura Fumio Matsuda Takayuki Tohge Keiko Yonekura‐Sakakibara Mami Yamazaki Kazuki Saito Issay Narumi 《The Plant journal : for cell and molecular biology》2010,62(4):549-559
Arabidopsis TRANSPARENT TESTA19 (TT19) encodes a glutathione‐S‐transferase (GST)‐like protein that is involved in the accumulation of proanthocyanidins (PAs) in the seed coat. PA accumulation sites in tt19 immature seeds were observed as small vacuolar‐like structures, whereas those in tt12, a mutant of the tonoplast‐bound transporter of PAs, and tt12 tt19 were observed at peripheral regions of small vacuoles. We found that tt19 immature seeds had small spherical structures showing unique thick morphology by differential interference contrast microscopy. The distribution pattern of the thick structures overlapped the location of PA accumulation sites, and the thick structures were outlined with GFP‐TT12 proteins in tt19. PA analysis showed higher (eightfold) levels of solvent‐insoluble PAs in tt19 immature seeds compared with the wild type. Metabolic profiling of the solvent‐soluble fraction by LC‐MS demonstrated that PA derivatives such as epicatechins and epicatechin oligomers, although highly accumulated in the wild type, were absent in tt19. We also revealed that tt12 specifically accumulated glycosylated epicatechins, the putative transport substrates for TT12. tt12 tt19 showed a similar metabolic profile to tt19. Given the cytosolic localization of functional GFP‐TT19 proteins, our results suggest that TT19, which acts prior to TT12, functions in the cytosol to maintain the regular accumulation of PA precursors, such as epicatechin and glycosylated epicatechin, in the vacuole. The PA pathway in the Arabidopsis seed coat is discussed in relation to the subcellular localization of PA metabolites. 相似文献
978.
Akie Homma Haruhiko Sato Tatsuya Tamura Akira Okamachi Takashi Emura Takenori Ishizawa Tatsuya Kato Tetsu Matsuura Shigeo Sato Yoshinobu Higuchi Tomoyuki Watanabe Hidetomo Kitamura Kentaro Asanuma Tadao Yamazaki Masahisa Ikemi Hironoshin Kitagawa Tadashi Morikawa Hitoshi Ikeya Kazuaki Maeda Koichi Takahashi Ryohchi Suzuki 《Bioorganic & medicinal chemistry》2010,18(3):1062-1075
We previously reported that a conjugate of hyaluronic acid (HA) and methotrexate (MTX) could be a prototype for future osteoarthritis drugs having the efficacy of the two clinically validated agents but with a reduced risk of the systemic side effects of MTX by using HA as the drug delivery carrier. To identify a clinical candidate, we attempted optimization of a lead, conjugate 1. Initially, in fragmentation experiments with cathepsins, we optimized the peptide part of HA–MTX conjugates to be simpler and more susceptible to enzymatic cleavage. Then we optimized the peptide, the linker, the molecular weight, and the binding ratio of the MTX of the conjugates to inhibit proliferation of human fibroblast-like synoviocytes in vitro and knee swelling in rat antigen-induced monoarthritis in vivo. Consequently, we found conjugate 30 (DK226) to be a candidate drug for the treatment of osteoarthritis. 相似文献
979.
Taiyo Toriba Takuya Suzaki Takahiro Yamaguchi Yoshihiro Ohmori Hirokazu Tsukaya Hiro-Yuki Hirano 《The Plant cell》2010,22(5):1452-1462
Establishment of adaxial-abaxial polarity is essential for lateral organ development. The mechanisms underlying the polarity establishment in the stamen remain unclear, whereas those in the leaf are well understood. Here, we investigated a rod-like lemma (rol) mutant of rice (Oryza sativa), in which the development of the stamen and lemma is severely compromised. We found that the rod-like structure of the lemma and disturbed anther patterning resulted from defects in the regulation of adaxial-abaxial polarity. Gene isolation indicated that the rol phenotype was caused by a weak mutation in SHOOTLESS2 (SHL2), which encodes an RNA-dependent RNA polymerase and functions in trans-acting small interfering RNA (ta-siRNA) production. Thus, ta-siRNA likely plays an important role in regulating the adaxial-abaxial polarity of floral organs in rice. Furthermore, we found that the spatial expression patterns of marker genes for adaxial-abaxial polarity are rearranged during anther development in the wild type. After this rearrangement, a newly formed polarity is likely to be established in a new developmental unit, the theca primordium. This idea is supported by observations of abnormal stamen development in the shl2-rol mutant. By contrast, the stamen filament is likely formed by abaxialization. Thus, a unique regulatory mechanism may be involved in regulating adaxial-abaxial polarity in stamen development. 相似文献
980.
Saori Kunii Koichi Morimoto Kouhei Nagai Takuya Saito Kenji Sato Ben'ichiro Tonomura 《The Journal of biological chemistry》2010,285(23):17465-17470
We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of α1 and α2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N- and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the α1 chain, pepsin cleaved between Asp1035 and Phe1036, and actinidain between Gly1032 and Gly1033. Thus, the actinidain-hydrolyzed α1 chain is shorter at the C terminus by three residues, Gly1033, Phe1034, and Asp1035. In the α2 chain, both proteases cleaved between Glu1030 and Val1031. We demonstrated that a synthetic nonapeptide mimicking the α1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the α1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation. 相似文献