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101.
Gene delivery to embryonic stem cells 总被引:1,自引:0,他引:1
Kobayashi N Rivas-Carrillo JD Soto-Gutierrez A Fukazawa T Chen Y Navarro-Alvarez N Tanaka N 《Birth defects research. Part C, Embryo today : reviews》2005,75(1):10-18
Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer). 相似文献
102.
Song T Hatano N Kume K Sugimoto K Yamaguchi F Tokuda M Watanabe Y 《FEBS letters》2005,579(25):5658-5662
We demonstrate that neuronal nitric-oxide synthase (nNOS) is directly inhibited through the phosphorylation of Thr(1296) in NG108-15 neuronal cells. Treatment of NG108-15 cells expressing nNOS with calyculin A, an inhibitor of protein phosphatase 1 and 2A, revealed a dose-dependent inhibition of nNOS enzyme activity with concomitant phosphorylation of Thr(1296) residue. Cells expressing a phosphorylation-deficient mutant in which Thr(1296) was changed to Ala proved resistant to phosphorylation and suppression of NOS activity. Mimicking phosphorylation mutant of nNOS in which Thr(1296) is changed to Asp showed a significant decrease in nNOS enzyme activity, being competitive with NADPH, relative to the wild-type enzyme. These data suggest that phosphorylation of nNOS at Thr(1296) may involve the attenuation of nitric oxide production in neuronal cells through the decrease of NADPH-binding to the enzyme. 相似文献
103.
Abe M Murai M Ichimaru N Kenmochi A Yoshida T Kubo A Kimura Y Moroda A Makabe H Nishioka T Miyoshi H 《Biochemistry》2005,44(45):14898-14906
Studies on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase), are useful for elucidating the structural and functional features of the terminal electron transfer step of this enzyme. Previous studies of the structure-activity relationship revealed that except for the alkyl spacer linking the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings), none of the multiple functional groups of these inhibitors is essential for potent inhibition. To elucidate the function of the alkyl spacer, two sets of systematically selected analogues were synthesized. First, the length of the spacer was varied widely. Second, the local flexibility of the spacer was specifically reduced by introducing multiple bond(s) into different regions of the spacer. The optimal length of the spacer for inhibition was approximately 13 carbon atoms. The decrease in the strength of the inhibitory effect caused by elongating the spacer from 13 carbons was much more drastic than that caused by shortening. Local flexibility in a specific region of the spacer was not important for the inhibition. These observations indicate that the active conformation of the spacer is not an extended form, and is not necessarily restricted to a certain rigid shape. Moreover, an analogue in which a spacer covering 10 carbon atoms was hardened into a rodlike shape still maintained a potent inhibitory effect. Our results strongly suggest that the spacer portion is free from steric congestion arising from the putative binding site probably because there is no cavity-like binding site for the spacer portion. The manner of acetogenin binding to the enzyme may not be explained by a simple "key and keyhole" analogy. 相似文献
104.
Wang JQ Kon J Mogi C Tobo M Damirin A Sato K Komachi M Malchinkhuu E Murata N Kimura T Kuwabara A Wakamatsu K Koizumi H Uede T Tsujimoto G Kurose H Sato T Harada A Misawa N Tomura H Okajima F 《The Journal of biological chemistry》2004,279(44):45626-45633
T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0-6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5'-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1- and GPR4-expressing cells but not for prostaglandin E(2)- and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pH-dependent action, although to a lesser extent. Psychosine-sensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against protein-sensing receptors, including TDAG8, GPR4, and OGR1. 相似文献
105.
Tokumitsu H Hatano N Inuzuka H Ishikawa Y Uyeda TQ Smith JL Kobayashi R 《The Journal of biological chemistry》2004,279(1):42-50
In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme. 相似文献
106.
Tamura Y Osuga J Adachi H Tozawa R Takanezawa Y Ohashi K Yahagi N Sekiya M Okazaki H Tomita S Iizuka Y Koizumi H Inaba T Yagyu H Kamada N Suzuki H Shimano H Kadowaki T Tsujimoto M Arai H Yamada N Ishibashi S 《The Journal of biological chemistry》2004,279(30):30938-30944
Scavenger receptor expressed by endothelial cells I (SREC-I) is a novel endocytic receptor for acetylated low density lipoprotein (LDL). Here we show that SREC-I is expressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly stimulated the expression of SREC-I in macrophages. In an initial attempt to clarify the role of SREC-I in the uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with a targeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming effect of the type A scavenger receptor (SR-A) on the uptake of Ac-LDL, we further generated mice lacking both SR-A and SREC-I (SR-A(-/-);SREC-I(-/-)) by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 90 and 6%, respectively. LPS increased the absolute contribution of SR-A and SREC-I by 1.9- and 2.3-fold, respectively. On the other hand, LPS decreased the absolute contribution of other pathways by 31%. Consistently, LPS did not increase the expression of other members of the scavenger receptor family such as CD36. In conclusion, SREC-I serves as a major endocytic receptor for Ac-LDL in LPS-stimulated macrophages lacking SR-A, suggesting that it has a key role in the development of atherosclerosis in concert with SR-A. 相似文献
107.
Specific inhibition of hepatitis C virus replication by cyclosporin A 总被引:13,自引:0,他引:13
Nakagawa M Sakamoto N Enomoto N Tanabe Y Kanazawa N Koyama T Kurosaki M Maekawa S Yamashiro T Chen CH Itsui Y Kakinuma S Watanabe M 《Biochemical and biophysical research communications》2004,313(1):42-47
The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection. 相似文献
108.
109.
Reiko Matsuyama Ichiro Takada Atsushi Yokoyama Sally Fujiyma-Nakamura Naoya Tsuji Hirochika Kitagawa Ryoji Fujiki Misun Kim Madoka Kouzu-Fujita Tetsu Yano Shigeaki Kato 《The Journal of biological chemistry》2010,285(24):18166-18176
Estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily and regulates many physiological functions, including mitochondrial biogenesis and lipid metabolism. ERRα enhances the transactivation function without endogenous ligand by associating with coactivators such as peroxisome proliferator-activated receptor γ coactivator 1 α and β (PGC-1α and -β) and members of the steroid receptor coactivator family. However, the molecular mechanism by which the transactivation function of ERRα is converted from a repressive state to an active state is poorly understood. Here we used biochemical purification techniques to identify ERRα-associated proteins in HeLa cells stably expressing ERRα. Interestingly, we found that double PHD fingers protein DPF2/BAF45d suppressed PGC-1α-dependent transactivation of ERRα by recognizing acetylated histone H3 and associating with HDAC1. DPF2 directly bound to ERRα and suppressed the transactivation function of nuclear receptors such as androgen receptor. DPF2 was recruited to ERR target gene promoters in myoblast cells, and knockdown of DPF2 derepressed the level of mRNA expressed by target genes of ERRα. These results show that DPF2 acts as a nuclear receptor-selective co-repressor for ERRα by associating with both acetylated histone H3 and HDAC1. 相似文献