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71.
Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca2+ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca2+-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca2+ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC50 = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC50 = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC50 = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension. 相似文献
72.
Kentaro M. Tanaka Aya Takahashi Naoyuki Fuse Toshiyuki Takano-Shimizu-Kouno 《Genetics》2014,197(2):739-742
Cell death is a mechanism utilized by organisms to eliminate excess cells during development. Here, we describe a novel regulator of caspase-independent cell death, Mabiki (Mabi), that is involved in the repair of the head patterning defects caused by extra copies of bicoid in Drosophila melanogaster. Mabiki functions together with caspase-dependent cell death mechanisms to provide robustness during development. 相似文献
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74.
Kenjiro Asagoshi Yuan Liu Aya Masaoka Li Lan Rajendra Prasad Julie K. Horton Ashley R. Brown Xiao-hong Wang Hussam M. Bdour Robert W. Sobol John-Stephen Taylor Akira Yasui Samuel H. Wilson 《DNA Repair》2010,9(2):109-119
We examined a role for DNA polymerase β (Pol β) in mammalian long patch base excision repair (LP BER). Although a role for Pol β is well known in single-nucleotide BER, information on this enzyme in the context of LP BER has been limited. To examine the question of Pol β involvement in LP BER, we made use of nucleotide excision repair-deficient human XPA cells expressing UVDE (XPA-UVDE), which introduces a nick directly 5′ to the cyclobutane pyrimidine dimer or 6-4 photoproduct, leaving ends with 3′-OH and 5′-phosphorylated UV lesion. We observed recruitment of GFP-fused Pol β to focal sites of nuclear UV irradiation, consistent with a role of Pol β in repair of UV-induced photoproducts adjacent to a strand break. This was the first evidence of Pol β recruitment in LP BER in vivo. In cell extract, a 5′-blocked oligodeoxynucleotide substrate containing a nicked 5′-cyclobutane pyrimidine dimer was repaired by Pol β-dependent LP BER. We also demonstrated Pol β involvement in LP BER by making use of mouse cells that are double null for XPA and Pol β. These results were extended by experiments with oligodeoxynucleotide substrates and purified human Pol β. 相似文献
75.
Kaoru Hida Giovanni Di Pasquale John A. Chiorini 《Archives of biochemistry and biophysics》2010,496(1):1-8
Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >105 members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands. 相似文献
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77.
Takezawa T Takeuchi T Nitani A Takayama Y Kino-Oka M Taya M Enosawa S 《Journal of biotechnology》2007,131(1):76-83
We previously succeeded in converting a soft and turbid disk of type-I collagen gel into a strong and transparent vitrigel membrane utilizing a concept for the vitrification of heat-denatured proteins and have demonstrated its protein-permeability and advantage as a scaffold for reconstructing crosstalk models between two different cell types. In this study, we observed the nano-structure of the type-I collagen vitrigel membrane and verified its utility for paracrine assays in vitro and drug delivery systems in vivo. Scanning electron microscopic observation revealed that the vitrigel membrane was a dense network architecture of typical type-I collagen fibrils. In the crosstalk model between PC-12 pheochromocytoma cells and L929 fibroblasts, nerve growth factor (NGF) secreted from L929 cells passed through the collagen vitrigel membrane and induced the neurite outgrowth of PC-12 cells by its paracrine effect. Also, the collagen vitrigel membrane containing vascular endothelial growth factor (VEGF) showed sustained-release of VEGF in vitro and its subcutaneous transplantation into a rat resulted in remarkable angiogenesis. These data suggest that the collagen vitrigel membrane is useful for paracrine assays in vitro and drug delivery systems in vivo. 相似文献
78.
Genome shuffling of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. U121 for improved production of hydroxycitric acid 总被引:2,自引:0,他引:2
(2S, 3R)-Hydroxycitric acid (HCA) from Hibiscus subdariffa inhibits pancreatic α-amylase and intestine α-glucosidase, leading to reduction of carbohydrate metabolism. In our previous
study, Streptomyces sp. U121 was identified as a producer of (2S, 3R)-HCA [Hida et al. (2005) Bioscience, Biotechnology, and Biochemistry 69:1555–1561]. Here, we applied genome shuffling of Streptomyces sp. U121 to achieve rapid improvement of HCA production. The initial mutant population was generated by nitrosoguanidine
treatment of the spores, and an improved population producing fivefold more HCA over wild type was obtained by three rounds
of genome shuffling. For efficient screening of the mutant library, trans-epoxyaconitic acid (EAA), an antibiotic analog of HCA, was utilized. EAA inhibited the regeneration of nonfused protoplasts,
resulting in selective screening of shuffled strains. Mutant strains with enhanced EAA resistance exhibited significantly
higher HCA production in liquid media. Furthermore, the best mutant showed increased cell growth in flask culture, as well
as increased HCA production. 相似文献
79.
FSAP (Factor VII-activating protease) can cleave and inactivate PDGF-BB (platelet-derived growth factor-BB) and thereby inhibits VSMC (vascular smooth-muscle cell) proliferation. The auto-activation of FSAP is facilitated by negatively charged polyanions such as heparin, dextransulfate or extracellular ribonucleic acids. Since auto-activation is essential for the anti-proliferative function of FSAP, the influence of nucleic acids as cofactors for the FSAP-mediated inhibition of PDGF-BB was investigated. Natural or artificial RNA was an effective cofactor for FSAP mediated PDGF-BB degradation, whereas the effect of DNA was weak. RNA-induced cleavage of PDGF-BB was inhibited by serine protease inhibitors. The pattern of PDGF-BB cleavage was identical with either heparin or RNA as a cofactor. One of the cleavage sites in PDGF-BB was at the positions 160-162 (R160KK162), which is an important region for receptor binding and activation. In VSMCs, PDGF-BB-stimulated DNA synthesis was inhibited by FSAP in the presence of RNA. RNA was more effective than DNA and the cofactor activity of RNA was neutralized after pretreatment with RNase. FSAP binding to RNA protected the nucleic acid from degradation by RNase. These data are relevant to situations where extracellular nucleic acids released from necrotic or apoptotic cells could activate local FSAP, leading to inhibition of PDGF-BB. 相似文献
80.