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71.
A phytase from Escherichia coli, AppA, has been the target of protein engineering to reduce the amount of undigested phosphates from livestock manure by making phosphorous from phytic acid available as a nutrient. To understand the contribution of each amino acid in the active site loop to the AppA activity, alanine and glycine scanning mutagenesis was undertaken. The results of phytase activity assay demonstrated loss of activity by mutations at charged residues within the conserved motif, supporting their importance in catalytic activity. In contrast, both conserved, non-polar residues and non-conserved residues tended to be tolerant to Ala and/or Gly mutations. Correlation analyses of chemical/structural characteristics of each mutation site against mutant activity revealed that the loop residues located closer to the substrate have greater contribution to the activity of AppA. These results may be useful in efficiently engineering AppA to improve its catalytic activity.
Abbreviations: AppA: pH 2.5 acid phosphatase; CSU: contacts of structural units; HAPs: histidine acid phosphatases; SASA: solvent accessible surface area; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SSM: site-saturation mutagenesis; WT: wild type 相似文献
72.
Yagi H Chuman Y Kozakai Y Imagawa T Takahashi Y Yoshimura F Tanino K Sakaguchi K 《Bioorganic & medicinal chemistry letters》2012,22(1):729-732
PPM1D is a p53-inducible Ser/Thr protein phosphatase. PPM1D gene amplification and overexpression have been reported in a variety of human tumors, including breast cancer and neuroblastoma. Because the phosphatase activity of PPM1D is essential for its oncogenic role, PPM1D inhibitors should be viable anti-cancer agents. In our current study, we showed that SPI-001 was a potent and specific PPM1D inhibitor. SPI-001 inhibited PPM1D phosphatase activity in PPM1D-overexpressing human breast cancer cells and increased phosphorylation of p53. Furthermore, SPI-001 suppressed cell proliferation by inducing apoptosis. Our present study suggested that SPI-001 was a potential lead compound in developing anti-cancer drugs. 相似文献
73.
Kumano-Kuramochi M Shimozu Y Wakita C Ohnishi-Kameyama M Shibata T Matsunaga S Takano-Ishikawa Y Watanabe J Goto M Xie Q Komba S Uchida K Machida S 《The Biochemical journal》2012,442(1):171-180
LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is an endothelial scavenger receptor that is important for the uptake of OxLDL (oxidized low-density lipoprotein) and contributes to the pathogenesis of atherosclerosis. However, the precise structural motifs of OxLDL that are recognized by LOX-1 are unknown. In the present study, we have identified products of lipid peroxidation of OxLDL that serve as ligands for LOX-1. We used CHO (Chinese-hamster ovary) cells that stably express LOX-1 to evaluate the ability of BSA modified by lipid peroxidation to compete with AcLDL (acetylated low-density lipoprotein). We found that HNE (4-hydroxy-2-nonenal)-modified proteins most potently inhibited the uptake of AcLDL. On the basis of the findings that HNE-modified BSA and oxidation of LDL resulted in the formation of HNE-histidine Michael adducts, we examined whether the HNE-histidine adducts could serve as ligands for LOX-1. The authentic HNE-histidine adduct inhibited the uptake of AcLDL in a dose-dependent manner. Furthermore, we found the interaction of LOX-1 with the HNE-histidine adduct to have a dissociation constant of 1.22×10(-8) M using a surface plasmon resonance assay. Finally, we showed that the HNE-histidine adduct stimulated the formation of reactive oxygen species and activated extracellular-signal-regulated kinase 1/2 and NF-κB (nuclear factor κB) in HAECs (human aortic endothelial cells); these signals initiate endothelial dysfunction and lead to atherosclerosis. The present study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL. 相似文献
74.
Paramecium species are extremely valuable organisms to enable experiments for the reestablishment of endosymbiosis. This is investigated in two different systems, the first with Paramecium caudatum and the endonuclear symbiotic bacterium Holospora species. Although most endosymbiotic bacteria cannot grow outside the host cell as a result of their reduced genome size, Holospora species can maintain their infectivity for a limited time. We found that an 89-kDa periplasmic protein has an important function for Holospora's invasion into the target nucleus, and that Holospora alters the host gene expression; the host thereby acquires resistance against various stresses. The second system is the symbiosis between P. bursaria and symbiotic Chlorella. Alga-free P. bursaria and the algae retain the ability to grow without a partner. Consequently, endosymbiosis between the aposymbiotic host cells and the symbiotic algae can be reestablished easily by mixing them. We now found four checkpoints for the reestablishment of the endosymbiosis between P. bursaria and the algae. The findings in the two systems provide excellent opportunities for us to elucidate not only infection processes but also to assess the associations leading to eukaryotic cell evolution. This paper summarizes recent progresses on reestablishment of the primary and the secondary endosymbiosis in Paramecium. 相似文献
75.
Impaired growth of pancreatic exocrine cells in transgenic mice expressing human activin betaE subunit 总被引:2,自引:0,他引:2
Hashimoto O Ushiro Y Sekiyama K Yamaguchi O Yoshioka K Mutoh K Hasegawa Y 《Biochemical and biophysical research communications》2006,341(2):416-424
Activins, TGF-beta superfamily members, have multiple functions in a variety of cells and tissues. Recently, additional activin beta subunit genes, betaC and betaE, have been identified. To explore the role of activin E, we created transgenic mice overexpressing human activin betaE subunit. There were pronounced differences in the pancreata of the transgenic animals as compared with their wild-type counterparts. Pancreatic weight, expressed relative to total body weight, was significantly reduced. Histologically, adipose replacement of acini in the exocrine pancreas was observed. There was a significant decrease in the number of PCNA-positive cells in the acinar cells, indicating reduced proliferation in the exocrine pancreas of the transgenic mice. However, quantitative pancreatic morphometry showed that the total number and mass of the islets of the transgenic mice were comparable with those of the nontransgenic control mice. Our findings suggest a role for activin E in regulating the proliferation of pancreatic exocrine cells. 相似文献
76.
Kikuko Ikeda Yuji NakayamaMayuko Ishii Yuuki ObataKousuke Kasahara Yasunori FukumotoNaoto Yamaguchi 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
The Src-family non-receptor-type tyrosine kinase Lyn, which is often associated with chemotherapeutic resistance in cancer, localizes not only to the plasma membrane but also Golgi membranes. Recently, we showed that Lyn, which is synthesized in the cytosol, is transported from the Golgi to the plasma membrane along the secretory pathway. However, it is still unclear how Golgi targeting of newly synthesized Lyn is regulated.Methods
Subcellular localization of Lyn and its mutants was determined by confocal microscopy.Results
We show that the kinase domain, but not the SH3 and SH2 domains, of Lyn is required for the targeting of Lyn to the Golgi, whereas the N-terminal lipids of the Lyn SH4 domain are not sufficient for its Golgi targeting. Although intact Lyn, which colocalizes with caveolin-positive Golgi membranes, can traffic toward the plasma membrane, kinase domain-deleted Lyn is immobilized on caveolin-negative Golgi membranes.General significance
Besides the SH4 domain, the Lyn kinase domain is important for targeting of newly synthesized Lyn to the Golgi, especially caveolin-positive transport membranes. Our results provide a novel role of the Lyn catalytic domain in the Golgi targeting of newly synthesized Lyn in a manner independent of its kinase activity. 相似文献77.
Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella. 相似文献
78.
Yasunori Fukumoto Yuuki Obata Kenichi Ishibashi Naoki Tamura Ikue Kikuchi Kazumasa Aoyama Yasuyuki Hattori Kunihiko Tsuda Yuji Nakayama Naoto Yamaguchi 《Cytotechnology》2010,62(1):73-82
Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although
transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to
experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000)
(PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient
delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and
intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important
for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C,
and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing
the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection
efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately
1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective,
high-efficiency transfection. 相似文献
79.
80.
Uno T Nakao A Masuda S Taniguchi Y Kanamaru K Yamagata H Nakamura M Imaishi H Oono K 《Journal of industrial microbiology & biotechnology》2006,33(12):1043-1050
We developed a system for bioconverting diverse compounds using P450s produced in Escherichia coli. Vectors for the expressing various P450 cDNAs quickly and easily in E. coli were developed by using several restriction enzyme sites. Three types of P450 (2C2, 2C29, and 2D22) were produced using these plasmids. Substrates were directly added to the incubation medium and metabolized. To obtain pure product from the medium, we first tried production of P450 in synthetic medium. The amount of another P450 2C43 produced in the synthetic medium was similar to the amount produced in Luria broth (LB) medium. Next, estradiol, a steroid, was added as a substrate, incubated, and the metabolite was extracted and analyzed by high-performance liquid chromatography. The metabolite extracted from synthetic medium was purer than that obtained from LB medium. Three P450s (2C29, 2C2, and 2A4) metabolized testosterone at different positions. P450 2C29 metabolized 7-ethoxycoumarin, androstendione, and dehydroepiandrosterone in this medium. P450s produced in the synthetic medium may be useful for producing various modified compounds for high-throughput screening. 相似文献