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181.
182.
Manabu Yamada Mitsuji Yamashita Takuya Suyama Junko Yamashita Kazuhide Asai Taishi Niimi Nobuhisa Ozaki Michio Fujie Kasthuraiah Maddali Satoki Nakamura Kazunori Ohnishi 《Bioorganic & medicinal chemistry letters》2010,20(19):5943-5946
4-Bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (3c) was first synthesized from 3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (2c) by a bromo-radical substitution reaction occurred at C-4 position by N-bromosuccinimide and 2,2′-azobisisobutyronitrile. The novel phospha sugar analogue 3c exerted high anti-proliferative effect on U937 cells evaluated by MTT in vitro methods and was much more efficient than that of Gleevec®, which is known as a molecule targeting chemotherapeutical agent. The substitution of 2-phospholenes at C-3 and C-4 position with methyl groups as well as 4-bromo substituent suggests a good anti-proliferative effect. 相似文献
183.
Claudin-16 is involved in the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle. Little is known about the mechanism regulating the tight junctional localization of claudin-16. Here, we examined the effect of Mg2+ deprivation on the distribution and function of claudin-16 using Madin-Darby canine kidney (MDCK) cells expressing FLAG-tagged claudin-16. Mg2+ deprivation inhibited the localization of claudin-16 at tight junctions, but did not affect the localization of other claudins. Re-addition of Mg2+ induced the tight junctional localization of claudin-16, which was inhibited by U0126, a MEK inhibitor. Transepithelial permeability to Mg2+ was also inhibited by U0126. The phosphorylation of ERK was reduced by Mg2+ deprivation, and recovered by re-addition of Mg2+. These results suggest that the MEK/ERK-dependent phosphorylation of claudin-16 affects the tight junctional localization and function of claudin-16. Mg2+ deprivation decreased the phosphothreonine levels of claudin-16. The phosphothreonine levels of T225A and T233A claudin-16 were decreased in the presence of Mg2+ and these mutants were widely distributed in the plasma membrane. Furthermore, TER and transepithelial Mg2+ permeability were decreased in the mutants. We suggest that the tight junctional localization of claudin-16 requires a physiological Mg2+ concentration and the phosphorylation of threonine residues via a MEK/ERK-dependent pathway. 相似文献
184.
Akira Ikari Kosuke Atomi Keishi Kinjo Yohei Sasaki Junko Sugatani 《Life sciences》2010,86(19-20):766-773
AimsLoss of magnesium (Mg2+) inhibits cell proliferation and augments nephrotoxicant-induced renal injury, but the role of Mg2+ has not been clarified in detail. We examined the effect of extracellular Mg2+ deprivation on a MEK–ERK cascade and cell proliferation using a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells.Main methodsMDCK cells were cultured in Mg2+-containing or Mg2+-free media. A HA-tagged constitutively active (CA)-MEK1 and a dominant negative (DN)-MEK1 were transfected into MDCK cells. The level of protein was examined by Western blotting. The intracellular free Mg2+ concentration ([Mg2+]i) was measured using a fluorescent dye, mag-fura 2. Cell proliferation was determined by WST-1 assay. Dead cells were identified by staining with annexin V-FITC and propidium iodide.Key findingsIn the presence of fetal calf serum (FCS), Mg2+ deprivation decreased phosphorylated-ERK1/2 (p-ERK1/2) levels and [Mg2+]i. Re-addition of Mg2+ increased p-ERK1/2 levels, which were inhibited by U0126, a specific inhibitor of a MEK–ERK cascade. Glutathione-S-transferase pull-down and coimmunoprecipitation assays showed that CA-MEK1 and DN-MEK1 binds with ERK1/2 in the presence of Mg2+. In contrast, neither CA-MEK1 nor DN-MEK1 bound to ERK1/2 in the absence of Mg2+. These results indicate that the MEK–ERK cascade is regulated by [Mg2+]i. Cell proliferation was increased by the treatment with FCS or the expression of CA-MEK1 in the presence of Mg2+, but was inhibited by Mg2+ deprivation. Mg2+ deprivation did not increase the number of dead cells.SignificanceMg2+ is involved in the regulation of the MEK–ERK cascade and cell proliferation in MDCK cells. 相似文献
185.
186.
Unlike known Chlamydomonas species, Chlamydomonas sp. strain W80, which was isolated from seawater, shows tolerance to salt and cadmium. In this study, we purified and characterized cysteine protease from Chlamydomonas sp. strain W80 cells and also investigated their response to oxidative stress. The protease was purified 2760-fold with a yield of 2.6% by five steps of successive chromatography. This protease had a pH optimum of 8.0 and was specific only for tert -butoxycarbonyl (Boc)-Leu-Arg-Arg-4-methylcoumaryl-7-amide (MCA) (Boc-LRR-MCA) and Boc-Val-Leu-Lys-MCA as substrates among eight fluorogenic peptides tested. The K m value was estimated to be 44.4 μ M for Boc-LRR-MCA. The molecular weight of the protease was determined to be approximately 102 kDa by Superdex 200 gel filtration and 60 kDa by SDS–PAGE, suggesting that this enzyme is a dimer. This enzyme was inhibited by the cysteine protease inhibitors leupeptin and N-ethylmaleimide but neither inhibited by phenylmethylsulfonyl fluoride or ethylenediaminetetraacetic acid nor activated by metal cations. These findings indicate that this enzyme is likely a cysteine protease. When strain W80 was grown under oxidative stress in the presence of methyl viologen and cadmium chloride, cysteine protease activity was about 30–90% higher than normal, whereas no changes were observed in carbon enrichment or senescence. It is likely that this protease is upregulated in response to oxidative stress and plays a role in the maintenance of cell metabolism under oxidative stress conditions. 相似文献
187.
188.
Ode H Matsuyama S Hata M Neya S Kakizawa J Sugiura W Hoshino T 《Journal of molecular biology》2007,370(3):598-607
A prominent characteristic of human immunodeficiency virus type 1 (HIV-1) is its high genetic variability, which generates diversity of the virus and often causes a serious problem of the emergence of drug-resistant mutants. Subtype B HIV-1 is dominant in advanced countries, and the mortality rate due to subtype B HIV-1 has been decreased during the past decade. In contrast, the number of patients with non-subtype B viruses is still increasing in developing countries. One of the reasons for the prevalence of non-subtype B viruses is lack of information about the biological and therapeutic differences between subtype B and non-subtype B viruses. M36I is the most frequently observed polymorphism in non-subtype B HIV-1 proteases. However, since the 36th residue is located at a non-active site of the protease and has no direct interaction with any ligands, the structural role of M36I remains unclear. Here, we performed molecular dynamics (MD) simulations of M36I protease in complex with nelfinavir and revealed the influence of the M36I mutation. The results show that M36I regulates the size of the binding cavity of the protease. The reason for the rare emergence of D30N variants in non-subtype B HIV-1 proteases was also clarified from our computational analysis. 相似文献
189.
In this paper, we describe an application of 202-membered fluorescently labeled peptide library designed to take an alpha-helix secondary structure. As a proof-of-concept experiment, a calmodulin (CaM)/calcineurin (Cn) pair was chosen to screen alpha-helical peptide ligands that tightly bind to CaM and also control enzymatic functions of Cn. Three peptides were successfully selected from the library by assaying Cn-phosphatase activities and peptide-CaM interactions (dual check process). The strategy using a designed peptide library shows real promise as a peptide-based high-throughput screening system. 相似文献
190.
Rowbottom MW Vickers TD Tamiya J Zhang M Dyck B Grey J Schwarz D Heise CE Hedrick M Wen J Tang H Wang H Fisher A Aparicio A Saunders J Goodfellow VS 《Bioorganic & medicinal chemistry letters》2007,17(8):2171-2178
The design, synthesis, and SAR of a series of substituted spirohydantoins are described. Optimization of an in-house screening hit gave compounds that exhibited potent binding affinity and functional activity at MCH-R1. 相似文献