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1.
We examined whether actin filaments are involved in the cAMP-dependent activation of a high affinity sodium/glucose cotransporter (SGLT1) using epithelial expression systems. The expression of enhanced green fluorescent protein-tagged SGLT1 (EGFP-SGLT1) in Madin-Darby canine kidney (MDCK) cells was revealed by Western blotting and confocal laser microscopy. 8-Br-cAMP, a membrane permeable cAMP analog, enhanced [14C]-α-methyl glucopyranoside ([14C]-AMG) uptake. Both basal and 8-Br-cAMP-elicited [14C]-AMG uptakes were inhibited by N-(2{[3-(4-bromophenyl)-2-propenyl]-amino}-ethyl)-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, and cytochalasin D, an actin filament formation inhibitor. Furthermore, cytochalasin D inhibited the distribution of EGFP-SGLT1 at the apical surface. These results suggest that the EGFP-SGLT1 protein is functionally expressed in the apical membrane of MDCK cells, and is up-regulated by a cAMP-dependent pathway requiring intact actin filaments. 相似文献
2.
Masaki Saito Hiroyuki Tanaka Masako Sasaki Hitoshi Kurose Norimichi Nakahata 《Cellular signalling》2010,22(1):41-46
The physiological role of the thromboxane A2 (TXA2) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA2 analogue. In the present study, we examined the detailed mechanisms of TXA2 receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na+/H+-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [3H]H2O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA2 receptor mediates water influx through aquaporins in astrocytoma cells via TXA2 receptor-mediated activation of Gα12/13, Rho A, Rho kinase and Na+/H+-exchanger. 相似文献
3.
Hitoshi Sawada Hideyoshi Yokosawa Shin-Ichi Ishii Motonori Hoshi 《Molecular reproduction and development》1982,5(3):291-301
The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl2. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH2Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH2Cl, and tosyl-Phe-CH2Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins. 相似文献
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Y Ishikawa K Kobayashi K Seki H Mizutani Y Kawasaki J Koike K Ijiri M Yamashita K Sugiura J Poynter T MacCallum G Anderson 《Biological Sciences in Space》1998,12(4):366-372
Several soluble components, peptidase and amino acids, and carbon isotopic ratio in the water retrieved from flight experiments of Autonomous Biological Systems (ABS) as well as ground control samples are analyzed to interpret the condition, dynamics, material balance of the ABS ecosystems. Organic carbons in flight samples were found to be more abundant compared with the control ones, which suggested the uniform ecosystems in low gravity might easily dissolve more soluble components. The Mir-1997 flight sample showed higher C/N ratio probably because of the dissolution of carbon-rich plant materials. 相似文献
6.
Ishige Fumiharu; Yamazaki Ken'ichi; Mori Hitoshi; Imaseki Hidemasa 《Plant & cell physiology》1991,32(5):681-690
The ethylene-induced synthesis and accumulation of proteinswere studied in the primary leaves of azuki bean (Vigna angularis).Seven different proteins, designated AZ17, 23, 27, 32, 35, 36,42 according to their molecular masses, were synthesized andaccumulated in response to ethylene. AZ27 and AZ42 were purifiedto homogeneity and characterized. AZ27 was identified as anacidic chitinase and accumulated in the extracellular space.The sequence of the 40 N-terminal amino acids of AZ27 showedno similarity to that of a basic chitinase from bean and tobacco,but it was highly homologous to that of a chitinase from virus-infectedcucumber leaves. AZ42 was identified as a glycoprotein thataccumulated intracellularly. A search for proteins with sequenceshomologous to an internal sequence of 18 amino acids in AZ42was unsuccessful. Immunochemical examination revealed that auxinand abscisic acid enhanced the ethylene-induced accumulationof AZ27 but not of AZ42. In contrast, levels of AZ42 were notaffected by auxin or abscisic acid, but cytokinin suppressedthe accumulation of one of the doublet bands of AZ42. TranslatablemRNAs coding for AZ27 and AZ42 were not present in leaves thathad not been treated with ethylene, but levels of these mRNAsincreased after such treatment. (Received March 1, 1991; Accepted May 8, 1991) 相似文献
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M Yamashita S Konagaya 《Comparative biochemistry and physiology. B, Comparative biochemistry》1990,96(2):247-252
1. Cathepsin L of the white muscle of chum salmon (Oncorhynchus keta) in spawning migration was purified to homogeneity by a series of chromatography on DEAE-Sephadex (1st), SP-Sephadex, CM-Sephadex, DEAE-Sephadex (2nd) and Sephadex G-100. 2. The molecular weight of salmon muscle cathepsin L was estimated to be 30,000 and its isoelectric point was 5.2. 3. Cathepsin L had a pH optimum of 5.6, required a thiol-reducing reagent for activation, and was inhibited by cysteine protease inhibitors. 4. The Km and kcat values for Z-Phe-Arg-MCA were determined to be 1.68 microM and 15.8 s-1, respectively. This enzyme hydrolyzed proteins such as insulin B chain, hemoglobin, serum albumin and azocasein easily. 5. The bond specificity to oxidized insulin B chain inferred that the enzyme had a preference for hydrophobic amino acid in P2 and P3 residues. 相似文献
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