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141.
Insulin-like growth factor-I stimulates diacylglycerol production via multiple pathways in Balb/c 3T3 cells 总被引:2,自引:0,他引:2
We previously reported that insulin-like growth factor-I (IGF-I) induced sustained calcium cycling across the plasma membrane in primed competent Balb/c 3T3 cells (Kojima, I., Matsunaga, H., Kurokawa, K., Ogata, E., and Nishimoto, I. (1989) J. Biol. Chem. 263, 16561-16567). The present study was conducted to examine whether IGF-I affected cellular metabolism of 1,2-diacylglycerol (1,2-DAG). In primed competent cells prelabeled with [3H]myristate, 1 nM IGF-I caused a 50% increase in [3H]DAG within 10 min. This increase in [3H]DAG was accompanied by 1) a decrease in radioactivity in the glycosylphosphatidylinositol fraction in [3H]glucosamine-labeled cells and a concomitant increase in [3H]inositol-glycan, and 2) a decrease in [3H]phosphatidylcholine and a concomitant elevation of [3H]phosphorylcholine in [3H]choline-labeled cells. When [3H]choline-labeled cells were treated with 10 nM 12-O-tetradecanoylphorbol-4-acetate (TPA), [3H]phosphatidylcholine was reduced by 50%. The TPA-induced reduction of [3H]phosphatidylcholine was completely blocked by 50 microM sphingosine and 50 microM H-7, inhibitors of protein kinase C. Both sphingosine and H-7 attenuated IGF-I-mediated reduction of [3H]phosphatidylcholine. In addition, treatment with IGF-I for 3 h or more resulted in sustained increase in 1,2-DAG mass, which was attenuated by cycloheximide. The increase in DAG mass was accompanied by enhanced incorporation of [14C]glucose into 1,2-DAG. These results indicate that, in primed competent Balb/c 3T3 cells, IGF-I stimulates 1,2-DAG production via multiple pathways and that IGF-I may induce breakdown of phosphatidylcholine by a mechanism involving protein kinase C. 相似文献
142.
Hasbaira Bolor Terumi Mori Sachie Nishiyama Yoshimasa Ito Eriko Hosoba Hidehito Inagaki Hiroshi Kogo Tamae Ohye Makiko Tsutsumi Takema Kato Maoqing Tong Haruki Nishizawa Kanako Pryor-Koishi Eri Kitaoka Tomio Sawada Yukio Nishiyama Yasuhiro Udagawa Hiroki Kurahashi 《American journal of human genetics》2009,84(1):14-20
143.
144.
SufC is an ATPase component of the SUF machinery, which is involved in the biosynthesis of Fe-S clusters. To gain insight into the function of this protein, we have determined the crystal structure of Escherichia coli SufC at 2.5A resolution. Despite the similarity of the overall structure with ABC-ATPases (nucleotide-binding domains of ABC transporters), some key differences were observed. Glu171, an invariant residue involved in ATP hydrolysis, is rotated away from the nucleotide-binding pocket to form a SufC-specific salt bridge with Lys152. Due to this salt bridge, D-loop that follows Glu171 is flipped out to the molecular surface, which may sterically inhibit the formation of an active dimer. Thus, the salt bridge may play a critical role in regulating ATPase activity and preventing wasteful ATP hydrolysis. Furthermore, SufC has a unique Q-loop structure on its surface, which may form a binding site for its partner proteins, SufB and/or SufD. 相似文献
145.
A simple protocol to introduce random mutations, named error-prone rolling circle amplification (RCA), is described. A template plasmid is amplified by RCA in the presence of MnCl2 and used for transformation of a host strain to give a mutant library with three to four random point mutations per kilobase throughout the entire plasmid. The prime advantage of this method is its simplicity. This protocol requires neither the design of specific primers nor the exploration of thermal cycling conditions. It takes just 10 min to prepare the reaction mixture, followed by overnight incubation and transformation of a host strain. This method permits rapid preparation of randomly mutated plasmid libraries, and will enable the wider adoption of random mutagenesis. 相似文献
146.
We report a method to prepare a DNA–enzyme conjugate using histidine-tag (His-tag) chemistry. A DNA oligonucleotide was modified with nitrilotriacetate (NTA), whose Kd was approximately 10?6 (M?1) toward a His-tag present on a recombinant protein via the complexation of Ni2+. His-tagged alkaline phosphatase (His-AP) was used as the model enzyme. Enzyme immobilization on the microplate revealed the conjugation of His-AP and the NTA-modified DNA via an Ni2+ complex. SPR measurements also proved the conjugation of His-AP with the NTA-modified DNA via an Ni2+ complex. The DNA–enzyme conjugate was then used for the detection of thrombin using a DNA aptamer. The DNA-AP conjugate successfully amplified the binding signal between the DNA aptamer and the thrombin, and the signal was measured as the fluorescent intensity derived from the AP-catalyzed reaction. The detection limit was 11 nM. Finally, we studied the effect of the release of the immobilized His-AP from the microplate on the AP activity, because the present strategy used a cleavable linker for the conjugation and the enzyme immobilization. The DNase-catalyzed release of the immobilized His-AP resulted in a 1.7-fold higher AP activity than observed when the His-AP was surface-immobilized. 相似文献
147.
Ishida-Kitagawa N Tanaka K Bao X Kimura T Miura T Kitaoka Y Hayashi K Sato M Maruoka M Ogawa T Miyoshi J Takeya T 《The Journal of biological chemistry》2012,287(21):17493-17502
Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles. In this study, we identified the gene encoding the lectin Siglec-15 as NFAT2-inducible, and we found that the protein product links RANK ligand-RANK-NFAT2 and DAP12 signaling in mouse osteoclasts. Both the recognition of sialylated glycans by the Siglec-15 V-set domain and the association with DAP12 through its Lys-272 are essential for its function. When Siglec-15 expression was knocked down, fewer multinucleated cells developed, and those that did were morphologically contracted with disordered actin-ring structures. These changes were accompanied by significantly reduced bone resorption. Siglec-15 formed complexes with Syk through DAP12 in response to vitronectin. Furthermore, chimeric molecules consisting of the extracellular and transmembrane regions of Siglec-15 with a K272A mutation and the cytoplasmic region of DAP12 significantly restored bone resorption in cells with knocked down Siglec-15 expression. Together, these results suggested that the Siglec-15-DAP12-Syk-signaling cascade plays a critical role in functional osteoclast formation. 相似文献
148.
Sakurama H Fushinobu S Hidaka M Yoshida E Honda Y Ashida H Kitaoka M Kumagai H Yamamoto K Katayama T 《The Journal of biological chemistry》2012,287(20):16709-16719
α-L-fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially α-L-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an α-L-fucosynthase that specifically introduces Le(a) and Le(x) antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Galβ1-3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an α-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-α-L-fucosidase (EC 3.2.1.111) and α-L-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Le(a/x) epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds. 相似文献
149.
Hiroyuki Tobita Akira Uemura Mitsutoshi Kitao Satoshi Kitaoka Hajime Utsugi 《Symbiosis (Philadelphia, Pa.)》2010,50(1-2):59-69
We examined the interactive effects of elevated CO2, soil phosphorus (P) availability, and soil drought on nodulation, nitrogenase activity, and biomass allocation in Alnus hirsuta and Alnus maximowiczii. Potted seedlings were grown in either ambient or elevated CO2 (36 Pa and 72 Pa CO2), with different levels of P (7.7 and 0.77 mgP pot?1 week?1 for high-P and low-P, respectively) and water supply in a natural daylight phytotron. Measurements of nitrogenase activity by an acetylene reduction assay failed to reveal significant effects of the treatments in any species. In high-P, nodule biomass increased under elevated CO2 and decreased under drought. In low-P, nodule biomass decreased substantially compared to high-P, but the effect of elevated CO2 on nodule biomass was unclear. Soil drought increased the partitioning of biomass into nodules, especially in A. hirsuta. These results suggest that with high P availability, elevated CO2 could promote N2 fixation by increasing nodule biomass even under drought. On the other hand, if soil P is limiting, elevated CO2 may not enhance N2 fixation because of the suppression of growth. 相似文献
150.
Florian A. Busch Jun Tominaga Masato Muroya Norihiko Shirakami Shunichi Takahashi Wataru Yamori Takuya Kitaoka Sara E. Milward Kohji Nishimura Erika Matsunami Yosuke Toda Chikako Higuchi Atsuko Muranaka Tsuneaki Takami Shunsuke Watanabe Toshinori Kinoshita Wataru Sakamoto Atsushi Sakamoto Hiroshi Shimada 《The Plant journal : for cell and molecular biology》2020,102(1):129-137
Bundle Sheath Defective 2, BSD2, is a stroma‐targeted protein initially identified as a factor required for the biogenesis of ribulose 1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) in maize. Plants and algae universally have a homologous gene for BSD2 and its deficiency causes a RuBisCO‐less phenotype. As RuBisCO can be the rate‐limiting step in CO2 assimilation, the overexpression of BSD2 might improve photosynthesis and productivity through the accumulation of RuBisCO. To examine this hypothesis, we produced BSD2 overexpression lines in Arabidopsis. Compared with wild type, the BSD2 overexpression lines BSD2ox‐2 and BSD2ox‐3 expressed 4.8‐fold and 8.8‐fold higher BSD2 mRNA, respectively, whereas the empty‐vector (EV) harbouring plants had a comparable expression level. The overexpression lines showed a significantly higher CO2 assimilation rate per available CO2 and productivity than EV plants. The maximum carboxylation rate per total catalytic site was accelerated in the overexpression lines, while the number of total catalytic sites and RuBisCO content were unaffected. We then isolated recombinant BSD2 (rBSD2) from E. coli and found that rBSD2 reduces disulfide bonds using reductants present in vivo, for example glutathione, and that rBSD2 has the ability to reactivate RuBisCO that has been inactivated by oxidants. Furthermore, 15% of RuBisCO freshly isolated from leaves of EV was oxidatively inactivated, as compared with 0% in BSD2‐overexpression lines, suggesting that the overexpression of BSD2 maintains RuBisCO to be in the reduced active form in vivo. Our results demonstrated that the overexpression of BSD2 improves photosynthetic efficiency in Arabidopsis and we conclude that it is involved in mediating RuBisCO activation. 相似文献