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641.
Thi Mai Hoa Bach Ryotaro Hara Kuniki Kino Iwao Ohtsu Nobuyuki Yoshida Hiroshi Takagi 《Applied microbiology and biotechnology》2013,97(1):247-257
The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP’s retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP. 相似文献
642.
Free radicals are reportedly involved in mucosal injury, including NH4OH-induced gastric lesions, but the kind, location and origin of radical generation have yet to be clarified. We developed the non-invasive measurement of reactive oxygen species (ROS) in stomach, and applied to mucosal injury. NH4OH-induced gastric lesions were prepared in rats, which were then given a nitroxyl probe intragastrically or intravenously, and the spectra of the gastric region were obtained by in vivo 300?MHz electron spin resonance (ESR) spectroscopy. The spectral change of the nitroxyl probe administered intragastrically was significantly enhanced 30?min after NH4OH administration, but no change occurred when the probe was given by intravenous injection. The enhanced change was confirmed to be due to ?OH generation, because it was completely suppressed by mannitol, catalase and desferrioxamine (DFO), and was not observed in neutropenic rats. NH4OH-induced neutrophil infiltration of the gastric mucosa was suppressed by intravenous injection of superoxide dismutase (SOD) or catalase, or by administration of allopurinol. The present study provided the direct evidence in NH4OH-treated living rats that ?OH produced from O2?- derived from neutrophils caused gastric lesion formation, while O2?- or H2O2 derived from the xanthine oxidase system in endothelial cells was involved in neutrophil infiltration. 相似文献
643.
Tomoko Kanno Eisuke F. Sato Shikibu Muranaka Hirofumi Fujita Takuzo Fujiwara Toshihiko Utsumi 《Free radical research》2013,47(1):27-35
Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca2+ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca2+ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca2+ increased the generation of H2O2 by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca2+ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca2+-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca2+ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c. 相似文献
644.
Dewiyani I. Widasari Yoshihiko Yano Takako Utsumi Didik S. Heriyanto Nungki Anggorowati Hanggoro T. Rinonce Totok Utoro Maria I. Lusida Soetjipto Widya Asmara Hak Hotta Yoshitake Hayashi 《Microbiology and immunology》2013,57(10):692-703
Hepatitis E is an emerging disease with a high incidence globally. Few data are available on hepatitis E virus (HEV) infection in Indonesia. To obtain molecular information on HEV infection in two regions of Indonesia with different customs and swine breeding conditions, serum samples from 137 swine farm workers, 100 blood donors and 100 swine (27 fecal samples also obtained) in Yogyakarta (Central Java) and from 12 and 64 swine farm workers, 42 and 135 local residents and 89 and 119 swine in Tulungagung (East Java) and Mengwi (Bali), respectively, from our previous study, were compared. Serological tests for anti‐HEV antibodies by ELISA, HEV‐RNA detection by RT‐PCR and phylogenetic analysis were performed. The total prevalence of anti‐HEV antibodies in humans was higher in Bali (11.6%) than in Java (5.1%; P = 0.015). No significant differences in anti‐HEV prevalence among swine farm workers and local residents in Java were found. The finding of swine HEV genotype 3 in specimens from Yogyakarta and genotype 4 from Tulungagung and Bali is somewhat different from other reports. We suggest other factors in addition to close contact with swine might play an important role in HEV transmission of non‐endemic/related custom groups. To the best of our knowledge, this is the first report on swine HEV genotype 3 in Indonesia. 相似文献
645.
Mahesh S. Chandak Takashi Nakamura Toshio Takenaka Tapan K. Chaudhuri Maho Yagi‐Utsumi Jin Chen Koichi Kato Kunihiro Kuwajima 《Protein science : a publication of the Protein Society》2013,22(4):486-491
Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D2O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D2O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride. 相似文献
646.
Background
Previous studies have reported the effects of statistics of luminance distribution on visual freshness perception using pictures which included the degradation process of food samples. However, these studies did not examine the effect of individual differences between the same kinds of food. Here we elucidate whether luminance distribution would continue to have a significant effect on visual freshness perception even if visual stimuli included individual differences in addition to the degradation process of foods.Methodology/principal findings
We took pictures of the degradation of three fishes over 3.29 hours in a controlled environment, then cropped square patches of their eyes from the original images as visual stimuli. Eleven participants performed paired comparison tests judging the visual freshness of the fish eyes at three points of degradation. Perceived freshness scores (PFS) were calculated using the Bradley-Terry Model for each image. The ANOVA revealed that the PFS for each fish decreased as the degradation time increased; however, the differences in the PFS between individual fish was larger for the shorter degradation time, and smaller for the longer degradation time. A multiple linear regression analysis was conducted in order to determine the relative importance of the statistics of luminance distribution of the stimulus images in predicting PFS. The results show that standard deviation and skewness in luminance distribution have a significant influence on PFS.Conclusions/significance
These results show that even if foodstuffs contain individual differences, visual freshness perception and changes in luminance distribution correlate with degradation time. 相似文献647.
Mitogen-activated protein kinases (MAP kinases) are intracellular signaling kinases activated by phosphorylation in response to a variety of extracellular stimuli. Mammalian MAP kinase pathways are composed of three major pathways: MEK1 (mitogen-activated protein kinase kinase 1)/ERK 1/2 (extracellular signal-regulated kinases 1/2)/p90 RSK (p90 ribosomal S6 kinase), JNK (c-Jun amino (N)-terminal kinase)/c-Jun, and p38 MAPK pathways. These pathways coordinately mediate physiological processes such as cell survival, protein synthesis, cell proliferation, growth, migration, and apoptosis. The involvement of MAP kinase in noise-induced hearing loss (NIHL) has been implicated in the cochlea; however, it is unknown how expression levels of MAP kinase change after the onset of NIHL and whether they are regulated by transient phosphorylation or protein synthesis. CBA/J mice were exposed to 120-dB octave band noise for 2 h. Auditory brainstem response confirmed a component of temporary threshold shift within 0–24 h and significant permanent threshold shift at 14 days after noise exposure. Levels and localizations of phospho- and total- MEK1/ERK1/2/p90 RSK, JNK/c-Jun, and p38 MAPK were comprehensively analyzed by the Bio-Plex® Suspension Array System and immunohistochemistry at 0, 3, 6, 12, 24 and 48 h after noise exposure. The phospho-MEK1/ERK1/2/p90 RSK signaling pathway was activated in the spiral ligament and the sensory and supporting cells of the organ of Corti, with peaks at 3–6 h and independently of regulations of total-MEK1/ERK1/2/p90 RSK. The expression of phospho-JNK and p38 MAPK showed late upregulation in spiral neurons at 48 h, in addition to early upregulations with peaks at 3 h after noise trauma. Phospho-p38 MAPK activation was dependent on upregulation of total-p38 MAPK. At present, comprehensive data on MAP kinase expression provide significant insight into understanding the molecular mechanism of NIHL, and for developing therapeutic models for acute sensorineural hearing loss. 相似文献
648.
649.
Nishizawa K Teraishi M Utsumi S Ishimoto M 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2007,114(4):755-764
Both α-amylase inhibitor-2 (αAI-2) and arcelin have been implicated in resistance of wild common bean (Phaseolus vulgaris L.) to the Mexican bean weevil (Zabrotes subfasciatus Boheman). Near isogenic lines (NILs) for arcelin 1–5 were generated by backcrossing wild common bean accessions with a cultivated
variety. Whereas seeds of a wild accession (G12953) containing both αAI-2 and arcelin 4 were completely resistant to Z. subfasciatus, those of the corresponding NIL were susceptible to infestation, suggesting that the principal determinant of resistance
was lost during backcrossing. Three independent lines of transgenic azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] expressing αAI-2 accumulated high levels of this protein in seeds. The expression of αAI-2 in these
lines conferred protection against the azuki bean weevil (Callosobruchus chinensis L.), likely through inhibition of larval digestive α-amylase. However, although the seed content of αAI-2 in these transgenic
lines was similar to that in a wild accession of common bean (G12953), it did not confer a level of resistance to Z. subfasciatus similar to that of the wild accession. These results suggest that αAI-2 alone does not provide a high level of resistance
to Z. subfasciatus. However, αAI-2 is an effective insecticidal protein with a spectrum of activity distinct from that of αAI-1, and it may
prove beneficial in genetic engineering of insect resistance in legumes. 相似文献
650.
Hashioka S Han YH Fujii S Kato T Monji A Utsumi H Sawada M Nakanishi H Kanba S 《Neurochemistry international》2007,50(3):499-506
Microglial activation and inflammatory processes have been implicated in the pathogenesis of a number of neurodegenerative disorders. Recently, peroxynitrite (ONOO(-)), the reaction product of superoxide (O(2)(-)) and nitric oxide (NO) both of which can be generated by activated microglia, has been demonstrated to act as a major mediator in the neurotoxicity induced by activated microglia. On the other hand, phospholipids such as phosphatidylserine (PS) and phosphatidylcholine (PC) have been reported to modulate the immune function of phagocytes. We therefore evaluated the effects of liposomes which comprise both PS and PC (PS/PC liposomes) or PC only (PC liposomes) regarding the production of both O(2)(-) and NO by lipopolysaccharide (LPS)/phorbol 12-myristate-13-acetate (PMA)-activated microglia using electron spin resonance (ESR) spin trap technique with a DEPMPO and Griess reaction, respectively. Pretreatment with PS/PC liposomes or PC liposomes considerably inhibited the signal intensity of O(2)(-) adduct associated with LPS/PMA-activated microglia in a dose-dependent manner. In addition, pretreatment with PS/PC liposomes also significantly reduced LPS/PMA-induced microglial NO production. In contrast, pretreatment with PC liposomes had no effect on the NO production. These results indicate that PS/PC liposomes can inhibit the microglial production of both NO and O(2)(-), and thus presumably prevent a subsequent formation of ONOO(-). Therefore, PS/PC liposomes appear to have both neuroprotective and anti-oxidative properties through the inhibition of microglial activation. 相似文献