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91.
Evidence has been presented showing two kinds of acidic protease activities in adult Schistosoma mansoni, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris suum. A haemoglobinolytic activity without adding any SH-containing compounds was maximal at pH 3.5, 2.5, 3.0 and 3.5 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The inhibitor studies demonstrated that this activity is ascribable to carboxyl protease(s). In the presence of dithiothreitol, activity on Azocoll (azo-dye coupled hide powder) was maximal at pH 4.6, 4.6, 3.5 and 5.6 in S. mansoni, D. immitis, Angiostrongylus cantonensis and Ascaris suum respectively. The effects of inhibitors demonstrated that this activity belongs to the thiol protease class. The intraspecific distribution of the protease activities was studied in some of the nematodes from which the organs could be anatomically separated. The distribution patterns of the haemoglobinolytic and azocollytic activities were quite different in An. cantonensis and much the same in As. suum. Based on the present results, acidic haemoglobinolytic activities reported in adult S. mansoni by other authors are thought to be due to carboxyl and thiol protease(s) respectively. 相似文献
92.
Keita Saito Miho Takahashi Masato Kamibayashi Toshihiko Ozawa 《Free radical research》2013,47(7):668-676
This study compared the superoxide detection abilities of four spin traps, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), 5-(diphenylphosphinoyl)-5-methyl-1pyrroline N-oxide (DPPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) in living cells. Electron spin resonance (ESR) signals of the superoxide adducts were observed when spin traps were added to a suspension of human oral polymorphonuclear leukocytes (OPMNs) stimulated by phorbol 12-myristate 13-acetate. The ESR signal of the CYPMPO-superoxide adduct (CYPMPO-OOH) increased for 24 min after the initiation of the reaction, whereas the signals from DMPO-OOH and DPPMPO-OOH peaked at 6 and 10 min, respectively. The maximum concentrations of DMPO-OOH, DPPMPO-OOH and CYPMPO-OOH in OPMNs were 1.9, 6.0 and 10.7 µM, respectively. Furthermore, CYPMPO could more efficiently trap superoxide in blood PMNs compared with DEPMPO. From these results, it was concluded that CYPMPO performs better than DMPO, DPPMPO and DEPMPO for superoxide measurements in living cell systems because it has lower cytotoxicity and its superoxide adduct has a longer lifetime. 相似文献
93.
Qing Feng Takeshi Kumagai Yasuyoshi Torii Yoshimasa Nakamura Toshihiko Osawa Koji Uchida 《Free radical research》2013,47(6):779-788
Oxidative stress has been implicated in the pathogenesis of numerous diseases, including cancer. In the present study, the protective effect of natural antioxidants, such as quercetin and tea polyphenols, on intracellular oxidative stress was studied. Here we report a novel function of quercetin and tea polyphenols, as potential inhibitors of 4-hydroxy-2-nonenal (HNE)-induced intracellular oxidative stress and cytotoxicity. In rat liver epithelial RL34 cells, a potent electrophile HNE dramatically induced the productions of reactive oxygen species (ROS), which correlated well with the reduction in cell viability. We found that quercetin and tea polyphenols, such as epigallocatechin gallate and theaflavins and their gallate esters, significantly inhibited the HNE-induced ROS production and cytotoxicity. In addition, HNE induced a transient decrease in the mitochondrial membrane potential (Δψ), which was also retarded by the antioxidants. These data suggest that the antioxidants, such as quercetin and tea polyphenols, are inhibitors against mitochondrial ROS production. 相似文献
94.
Chiho Nishizawa Keizo Takeshita Jun-ichi Ueda Michiko Mizuno Kazuo T. Suzuki Toshihiko Ozawa 《Free radical research》2013,47(4):385-392
Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (1O2) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of 1O2 with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from 1O2-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from 1O2, and that spin trap-mediated ?OH generation hardly occurs with DEPMPO. 相似文献
95.
Koko Moriya Kei Nagatoshi Yoshimi Noriyasu Tsuyoshi Okamura Emi Takamitsu Takashi Suzuki Toshihiko Utsumi 《PloS one》2013,8(11)
N-myristoylation of eukaryotic cellular proteins has been recognized as a modification that occurs mainly on cytoplasmic proteins. In this study, we examined the membrane localization, membrane integration, and intracellular localization of four recently identified human N-myristoylated proteins with predicted transmembrane domains. As a result, it was found that protein Lunapark, the human ortholog of yeast protein Lnp1p that has recently been found to be involved in network formation of the endoplasmic reticulum (ER), is an N-myristoylated polytopic integral membrane protein. Analysis of tumor necrosis factor-fusion proteins with each of the two putative transmembrane domains and their flanking regions of protein Lunapark revealed that transmembrane domain 1 and 2 functioned as type II signal anchor sequence and stop transfer sequence, respectively, and together generated a double-spanning integral membrane protein with an N-/C-terminal cytoplasmic orientation. Immunofluorescence staining of HEK293T cells transfected with a cDNA encoding protein Lunapark tagged with FLAG-tag at its C-terminus revealed that overexpressed protein Lunapark localized mainly to the peripheral ER and induced the formation of large polygonal tubular structures. Morphological changes in the ER induced by overexpressed protein Lunapark were significantly inhibited by the inhibition of protein N-myristoylation by means of replacing Gly2 with Ala. These results indicated that protein N-myristoylation plays a critical role in the ER morphological change induced by overexpression of protein Lunapark. 相似文献
96.
Kazuyo Yasuda Toshihiko Torigoe Rena Morita Takahumi Kuroda Akari Takahashi Junichi Matsuzaki Vitaly Kochin Hiroko Asanuma Tadashi Hasegawa Tsuyoshi Saito Yoshihiko Hirohashi Noriyuki Sato 《PloS one》2013,8(8)
Cancer stem-like cells (CSCs)/cancer-initiaiting cells (CICs) are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP) analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDHBr) cells from ovarian cancer cells. Both SP cells and ALDHBr cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2), than those of main population (MP) cells and ALDHLow cells, respectively. We analyzed an SP and ALDHBr overlapping population (SP/ALDHBr), and the SP/ALDHBr population exhibited higher tumor-initiating ability than that of SP cells or ALDHBr cells, enabling initiation of tumor with as few as 102 cells. Furthermore, SP/ADLHBr population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDHLow, MP/ALDHBr and MP/ALDHLow cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDHBr population was detected in several gynecological cancer cells with ratios of 0.1% for HEC—1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDHBr overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs. 相似文献
97.
Hisato Kobayashi Eikichi Yanagisawa Akihiko Sakashita Naoko Sugawara Shiori Kumakura Hidehiko Ogawa Hidenori Akutsu Kenichiro Hata Kazuhiko Nakabayashi Tomohiro Kono 《Epigenetics》2013,8(6):635-651
Artificial induction of active DNA demethylation appears to be a possible and useful strategy in molecular biology research and therapy development. Dimethyl sulfoxide (DMSO) was shown to cause phenotypic changes in embryonic stem cells altering the genome-wide DNA methylation profiles. Here we report that DMSO increases global and gene-specific DNA hydroxymethylation levels in pre-osteoblastic MC3T3-E1 cells. After 1 day, DMSO increased the expression of genes involved in DNA hydroxymethylation (TET) and nucleotide excision repair (GADD45) and decreased the expression of genes related to DNA methylation (Dnmt1, Dnmt3b, Hells). Already 12 hours after seeding, before first replication, DMSO increased the expression of the pro-apoptotic gene Fas and of the early osteoblastic factor Dlx5, which proved to be Tet1 dependent. At this time an increase of 5-methyl-cytosine hydroxylation (5-hmC) with a concomitant loss of methyl-cytosines on Fas and Dlx5 promoters as well as an increase in global 5-hmC and loss in global DNA methylation was observed. Time course-staining of nuclei suggested euchromatic localization of DMSO induced 5-hmC. As consequence of induced Fas expression, caspase 3/7 and 8 activities were increased indicating apoptosis. After 5 days, the effect of DMSO on promoter- and global methylation as well as on gene expression of Fas and Dlx5 and on caspases activities was reduced or reversed indicating down-regulation of apoptosis. At this time, up regulation of genes important for matrix synthesis suggests that DMSO via hydroxymethylation of the Fas promoter initially stimulates apoptosis in a subpopulation of the heterogeneous MC3T3-E1 cell line, leaving a cell population of extra-cellular matrix producing osteoblasts. 相似文献
98.
Takeo Kawahara Naoe Hotta Yukiko Ozawa Seiichi Kato Keiko Kano Yukihiro Yokoyama Masato Nagino Takashi Takahashi Kiyoshi Yanagisawa 《PloS one》2013,8(12)
Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies. 相似文献
99.
More than 150 types of Human papillomaviruses (HPVs) have been isolated from numerous cutaneous and/or mucosal lesions. Flat wart samples on the face from 36 immunocompetent patients were collected and screened for HPV. From one sample, we cloned a putative novel genotype. The novel type consisted of 7779 bp in length with a GC content of 47.1%, containing open reading frames for putative early proteins (E1, E2, E4, E6, and E7) and two late proteins (L1 and L2). Homology searches and phylogenetic analyses indicated that it belonged to Alphapapillomavirus (Alpha-PV) species 2 and most closely resembled HPV 3. The virus fulfilled the definition of a novel type, and was named HPV 160 by the Reference Center for Papillomaviruses. The putative E7 protein of HPV 160 as well as HPV 29, 77, and 78 contained the Leu-X-Cys-X-Glu pRB-binding motif but other Alpha-PV species 2 (HPV 3, 10, 28, 94, 117, and 125) did not have this conserved motif. 相似文献
100.
Yanagisawa Takahiro Ishii Masakazu Takahashi Manami Fujishima Kei Nishimura Masahiro 《Molecular biology reports》2020,47(9):6841-6854
Molecular Biology Reports - LL-37, the only member of the cathelicidin family of cationic antimicrobial peptides in humans has been shown to exhibit a wide variety of biological actions in addition... 相似文献