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981.
Kuwasako K Kitamura K Nagata S Kato J 《Biochemical and biophysical research communications》2008,377(1):109-113
Receptor activity-modifying protein (RAMP)-2 and -3 chaperone calcitonin receptor-like receptor (CRLR) to the plasma membrane, where together they form heterodimeric adrenomedullin (AM) receptors. We investigated the contributions made by His residues situated in the RAMP extracellular domain to AM receptor trafficking and receptor signaling by co-expressing hCRLR and V5-tagged-hRAMP2 or -3 mutants in which a His residue was substituted with Ala in HEK-293 cells. Flow cytometric analysis revealed that hRAMP2-H71A mediated normal hCRLR surface delivery, but the resultant heterodimers showed significantly diminished [125I]AM binding and AM-evoked cAMP production. Expression of hRAMP2-H124A and -H127A impaired surface delivery of hCRLR, which impaired or abolishing AM binding and receptor signaling. Although hRAMP3-H97A mediated full surface delivery of hCRLR, the resultant heterodimers showed impaired AM binding and signaling. Other His residues appeared uninvolved in hCRLR-related functions. Thus, the His residues of hRAMP2 and -3 differentially govern AM receptor function. 相似文献
982.
Sakamoto E Tsukioka S Oie S Kobunai T Tsujimoto H Sakamoto K Okayama Y Sugimoto Y Oka T Fukushima M Oka T 《Biochemical and biophysical research communications》2008,365(4):801-807
Although 5-fluorouracil (5-FU) plus leucovorin (LV) is a standard chemotherapy regimen for colorectal cancer, the factors that determine the LV-mediated enhancement of the antitumor activity of 5-FU have remained unknown. We investigated the roles of folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH), which are the main enzymes involved in folate metabolism, in the effect of LV. LV enhanced the anticancer activity of 5-FU and the level of reduced folate in human colon cancer cells. Small-interfering RNA (siRNA) transfected into DLD-1 cells to downregulate FPGS reduced the basal level of reduced folate, the folate level after LV treatment, and the enhancement of 5-fluoro-2′-deoxyuridine (FdUrd)-induced cytotoxicity elicited by LV. By contrast, the downregulation of GGH by siRNA increased cellular sensitivity to FdUrd combined with LV. These results suggest that FPGS and GGH expression levels in tumors are determinants of the efficacy of LV in enhancing the antitumor activity of 5-FU. 相似文献
983.
984.
Uemura S Kurose T Suzuki T Yoshida S Ito M Saito M Horiuchi M Inagaki F Igarashi Y Inokuchi J 《Glycobiology》2006,16(3):258-270
The sialyltranferase ST3Gal-V transfers a sialic acid to lactosylceramide. We investigated the role of each of the N-glycans modifying mouse ST3Gal-V (mST3Gal-V) by measuring the in vitro enzyme activity of Chinese hamster ovary (CHO) cells transfected with ST3Gal-V cDNA or its mutants. By examining mutants of mST3Gal-V, in which each asparagine was replaced with glutamine (N180Q, N224Q, N334Q), we determined that all three sites are N-glycosylated and that each N-glycan is required for enzyme activity. Despite their importance, N-glycosylation sites in ST3Gal-V are not conserved among species. Therefore, we considered whether the function in the activity that is performed in mST3Gal-V by the N-glycan could be substituted for by specific amino acid residues selected from the ST3Gal-V of other species or from related sialyltransferases (ST3Gal-I, -II, -III, and -IV), placed at or near the glycosylation sites. To this end, we constructed a series of interspecies mutants for mST3Gal-V, specifically, mST3Gal-V-H177D-N180S (medaka or tetraodon type), mST3Gal-V-N224K (human type), and mST3Gal-V-T336Q (zebrafish type). The ST3Gal-V activity of these mutants was quite similar to that of the wild-type enzyme. Thus, we have demonstrated here that the N-glycans on mST3Gal-V are required for activity but can be substituted for specific amino acid residues placed at or near the glycosylation sites. We named this method SUNGA (substitution of N-glycan functions in glycosyltransferases by specific amino acids). Furthermore, we verified that the ST3Gal-V mutant created using the SUNGA method maintains its high activity when expressed in Escherichia coli thereby establishing the usefulness of the SUNGA method in exploring the function of N-glycans in vivo. 相似文献
985.
Kitagawa M Moriyama T Ito H Ozasa S Adachi A Yasuda J Ookura T Inakuma T Kasumi T Ishiguro Y Ito Y 《Bioscience, biotechnology, and biochemistry》2006,70(5):1227-1233
The ripening inhibitor (rin) mutant tomato yields non-ripening fruit, and the rin hybrid fruit (RIN/rin) shows an intermediate phenotype between the wild and mutant fruit, that is, red-ripe and extended shelf life. We found by a microarray analysis that the genes encoding possible allergenic proteins were expressed at a significantly lower level in the rin hybrid fruit than in the wild-type fruit. These allergenic proteins, which were beta-fructofuranosidase and polygalacturonase 2A (PG-2A), were confirmed to accumulate at a lower level in the rin hybrid fruit than in the wild-type fruit. The immunoglobulin E (IgE) in serum from a tomato-allergic patient showed lower reactivity to the extract of the rin hybrid fruit than to that of the wild fruit. These results suggest that the rin gene has the potential to regulate allergen accumulation in tomato fruit. 相似文献
986.
The polarized synthesis of cell wall components such as chitin is essential for the hyphal tip growth of filamentous fungi. The actin cytoskeleton is known to play important roles in the determination of hyphal polarity in Aspergillus nidulans. Previously, we suggested that CsmA, a chitin synthase with a myosin motor-like domain (MMD), was involved in polarized chitin synthesis in a manner dependent on the interaction between the MMD and the actin cytoskeleton. The genome database indicates that A. nidulans possesses another gene encoding another chitin synthase with an MMD. In this study, we characterized this gene, which we designated csmB. The csmB null mutants examined were viable, although they exhibited defective phenotypes, including the formation of balloons and intrahyphal hyphae and the lysis of subapical regions, which were similar to those obtained with csmA null mutants. Moreover, csmA csmB double null mutants were not viable. Mutants in which csmB was deleted and the expression of csmA was under the control of the alcA promoter were viable but severely impaired in terms of hyphal growth under alcA-repressing conditions. We revealed that CsmB with three copies of a FLAG epitope tag localized at the hyphal tips and forming septa, and that the MMD of CsmB was able to bind to actin filaments in vitro. These results suggest that CsmA and CsmB perform compensatory functions that are essential for hyphal tip growth. 相似文献
987.
Kouji Kobiyama Fumihiko Takeshita Nao Jounai Asako Sakaue-Sawano Atsushi Miyawaki Ken J. Ishii Taro Kawai Shin Sasaki Hisashi Hirano Norihisa Ishii Kenji Okuda Koichi Suzuki 《Journal of virology》2010,84(2):822-832
Fragments of double-stranded DNA (dsDNA) forming a right-handed helical structure (B-DNA) stimulate cells to produce type I interferons (IFNs). While an adaptor molecule, IFN-β promoter stimulator 1 (IPS-1), mediates dsDNA-induced cellular signaling in human cells, the underlying molecular mechanism is not fully understood. Here, we demonstrate that the extrachromosomal histone H2B mediates innate antiviral immune responses in human cells. H2B physically interacts with IPS-1 through the association with a newly identified adaptor, CIAO (COOH-terminal importin 9-related adaptor organizing histone H2B and IPS-1), to transmit the cellular signaling for dsDNA but not immunostimulatory RNA. Extrachromosomal histone H2B was biologically crucial for cell-autonomous responses to protect against multiplication of DNA viruses but not an RNA virus. Thus, the present findings provide evidence indicating that the extrachromosomal histone H2B is engaged in the signaling pathway initiated by dsDNA to trigger antiviral innate immune responses.Fragments of nucleic acids derived from either infectious agents or host cells activate cell-autonomous responses to inhibit multiplication of certain viruses by inducing type I interferon (IFN) production (5). Such effects are more evident when double-stranded DNA (dsDNA) is transduced into the intracellular compartment by use of a transfection agent or electroporation method, suggesting that the DNA sensing system recognizes aberrant DNA fragments inside the cell (6, 21, 23). dsDNA forming a right-handed helical structure, i.e., B-DNA, has a greater ability to induce type I IFNs than Z-DNA, which has a left-handed zig-zag structure (6). dsDNA activates type I IFN production in a wide variety of cell types, including immune cells, such as dendritic cells and macrophages, and nonimmune cells, such as fibroblasts, epithelial cells, and thyroid cells (6, 23). Such effects of dsDNA were corroborated by the observation in mice deficient for DNase II, in which intracellular accumulation of undegraded DNA fragments resulted in hyperproduction of IFN-β, dysregulation of erythropoiesis, and symptoms resembling rheumatoid arthritis (12, 28). The loss-of-function mutation of the DNase I gene has been found in patients with systemic lupus erythematosus (SLE) and, in fact, DNase I−/− mice manifest SLE-like symptoms with anti-DNA antibody (Ab) production (18, 27).The immunostimulatory property of dsDNA is quite similar to that of immunostimulatory RNA (isRNA), such as dsRNA and 5′-triphosphate RNA (2, 6). Indeed, the signaling pathways engaged by dsDNA in part are shared with those for isRNA. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) directly associate with isRNA and trigger signaling, while it has been demonstrated that RIG-I does not directly interact with dsDNA but mediates its signaling in a human hepatoma cell line, Huh7 (2). IFN-β promoter stimulator 1 (IPS-1, also known as mitochondrial antiviral signaling), mediates the downstream signaling induced by dsDNA or isRNA in humans, while IPS-1 solely mediates isRNA but not dsDNA signaling in mice (2, 6, 15, 22). In contrast, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi) are essential for dsDNA- or isRNA-induced type I IFN production in both humans and mice (2, 6). While examining distinct molecules involved in dsDNA-mediated but not isRNA-mediated upstream signaling, Z-DNA binding protein 1 (ZBP1, also known as DNA-dependent activator of IFN regulatory factors [DAI]) was identified as a candidate cytosolic DNA sensor, at least in a mouse connective tissue cell line, L929, although its in vivo role was dispensable (7, 24, 26). Recently, a PYHIN family member, Absent in melanoma 2 (AIM2) protein, was shown to associate with an inflammasome signaling adaptor, apoptosis-associated speck-like protein containing a CARD (ASC), and to play a critical role for caspase 1 activation and interleukin-1β (IL-1β) secretion in response to dsDNA (1, 3, 4, 20).In the present study, we show that extrachromosomal histone H2B is responsible for the dsDNA-induced type I IFN production in human cells and for the innate immune response to DNA virus infection. 相似文献
988.
989.
Yoshikazu Shimoda Hisayuki Mitsui Hiroko Kamimatsuse Kiwamu Minamisawa Eri Nishiyama Yoshiyuki Ohtsubo Yuji Nagata Masataka Tsuda Sayaka Shinpo Akiko Watanabe Mitsuyo Kohara Manabu Yamada Yasukazu Nakamura Satoshi Tabata Shusei Sato 《DNA research》2008,15(5):297-308
Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology.Key words: Mesorhizobium loti, signature-tagged mutagenesis, mutant library, reverse genetics 相似文献
990.
Hakamada S Sonoyama T Ichiki S Nakamura S Uchiyama S Kobayashi Y Sambongi Y 《Bioscience, biotechnology, and biochemistry》2008,72(8):2103-2109
Cytochrome c(552) (PH c(552)) from moderately thermophilic Hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome c(552) (HT c(552)) from thermophilic Hydrogenobacter thermophilus and cytochrome c(551) (PA c(551)) from mesophilic Pseudomonas aeruginosa. To understand the mechanism of stabilization of PH c(552), we introduced mutations into PH c(552) at five sites, which, in HT c(552), are occupied by the amino acids responsible for stability higher than the less stable PA c(551). When PH c(552) Val-78 was mutated to Ile, as found in HT c(552), the resulting variant showed increased stability. Mutation of Ala-7, Met-13, and Tyr-34 to the corresponding residues in PA c(551) (Phe, Val, and Phe, respectively) resulted in destabilization. We also found that PH c(552) Lys-43 contributed to stability through the formation of an attractive electrostatic interaction with Asp-39. These results suggest that the intermediate stability of PH c(552) is due to the amino acids at these five sites. 相似文献