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61.
Manabu Yamamoto Satoshi Okumura Carsten Schwencke Junichi Sadoshima Yoshihiro Ishikawa 《The Histochemical journal》1999,31(4):241-243
A high efficiency transfection protocol employing a common polycationic lipid is described. Using LipofectAMINE, a widely used transfection reagent, we transfected 293T cells with a plasmid harboring the -galactosidase (-gal) gene. The transfection efficiency was determined by direct staining for X-gal. The conventional transfection protocol achieved an efficiency of <40% while our protocol, which employs the repetition of transfection a few times, achieved an efficiency of approximately 80%. Thus, a dramatic increase in transfection efficiency can be obtained by simply repeating transfection with the use of a common polycationic lipid. This method will be useful in many molecular biological experiments. 相似文献
62.
63.
Tsukamoto H Hishinuma T Suzuki N Tayama R Hiratsuka M Yoshihisa T Mizugaki M Goto J 《Prostaglandins & other lipid mediators》2004,73(3-4):191-213
Thiazolidinedione, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, has been used as an anti-diabetic drug and as an useful tool to elucidate multiple PPARgamma functions by in vitro and in vivo studies. We investigated the effects of thiazolidinediones on prostanoid production in lipopolysaccharide-stimulated cells. The high concentrations (>10 microM) of rosiglitazone and pioglitazone significantly increased lipopolysaccharide-stimulated prostanoid production such as thromboxane A2 and prostaglandin E2. However, PPARgamma antagonist could not inhibit them. In PPARgamma-deficient cells, thiazolidinediones increased prostaglandin E2 production. Thiazolidinediones increased arachidonic acid (AA) release from the cell membrane by not stimulating AA releasing process involving several phospholipase A2s but inhibiting AA reuptaking process. The expression of cyclooxygenase-1 and cyclooxygenase-2 were not affected by thiazolidinediones. In this study, we demonstrated that high concentrations of TZDs increased AA release by the inhibition of AA reuptaking process, leading to subsequent increase in the prostanoid production in a PPARgamma-independent manner. This mechanism provides useful information for the elucidation of multiple PPARgamma functions and diabetic drug therapy. 相似文献
64.
Paik MJ Lee Y Goto J Kim KR 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,803(2):257-265
Profens were converted into diastereomeric (R)-(+)-1-phenylethylamides using ethyl chloroformate and triethylamine in dichloromethane. Gas chromatographic analysis on dual-columns with different polarities provided complete enantioresolution of eight profens, facilitating chiral discrimination based on matching with retention index sets characteristic of each enantiomer. The present method was linear (r >/= 0.9992) with good precision (0.8-6.0%) and accuracy (-9.3 to 0.003%), allowing detection of trace (R)-profens in optical purity test on four (S)-profen mixture in a single run. And the method allowed simultaneous enantiomeric screening for ibuprofen enantiomers and their chiral metabolites excreted in urine following administration of racemic ibuprofen. 相似文献
65.
Nitric oxide inhibits ultraviolet B-induced murine keratinocyte apoptosis by regulating apoptotic signaling cascades 总被引:2,自引:0,他引:2
Cytotoxic effects of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) are considered to be one of the major causes of inflammatory diseases. On the other hand, protective effects of NO on toxic insults-induced cellular damage/apoptosis have been demonstrated recently. Ultraviolet B (UVB)-induced apoptosis of epidermal keratinocytes leads to skin inflammation and photoageing. However, it has not been elucidated what kind of effects NO has on UVB-induced keratinocyte apoptosis. Thus, in the present study, we investigated the problem and demonstrated that NO from NO donor suppressed UVB-induced apoptosis of murine keratinocytes. In addition, NO significantly suppressed activities of caspase 3, caspase 8 and caspase 9 that had been upregulated by UVB radiation. NO also suppressed p53 expression that had been upregulated by UVB radiation and upregulated Bcl-2 expression that had been downregulated by UVB radiation. These findings suggested that NO might suppress UVB-induced keratinocyte apoptosis by regulating apoptotic signaling cascades in p53, Bcl-2, caspase3, caspase 8 and caspase 9. 相似文献
66.
Grishko V Pastukh V Solodushko V Gillespie M Azuma J Schaffer S 《American journal of physiology. Heart and circulatory physiology》2003,285(6):H2364-H2372
Angiotensin II contributes to ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; however, the mechanism underlying the latter phenomenon is poorly understood. One possibility that has been advanced is that angiotensin II activates NADPH oxidase, generating free radicals that trigger apoptosis. In apparent support of this notion, it was found that angiotensin II-mediated apoptosis in the cardiomyocyte is blocked by the NADPH oxidase inhibitor diphenylene iodonium. However, three lines of evidence suggest that peroxynitrite, rather than superoxide, is responsible for angiotensin II-mediated DNA damage and apoptosis. First, the inducible nitric oxide inhibitor aminoguanidine prevents angiotensin II-induced DNA damage and apoptosis. Second, based on ligation-mediated PCR, the pattern of angiotensin II-induced DNA damage resembles peroxynitritemediated damage rather than damage caused by either superoxide or nitric oxide. Third, angiotensin II activates p53 through the phosphorylation of Ser15 and Ser20, residues that are commonly phosphorylated in response to DNA damage. It is proposed that angiotensin II promotes the oxidation of DNA, which in turn activates p53 to mediate apoptosis. 相似文献
67.
Free-energy landscape of a chameleon sequence in explicit water and its inherent alpha/beta bifacial property 下载免费PDF全文
A sequence in yeast MATalpha2/MCM1/DNA complex that folds into alpha-helix or beta-hairpin depending on the surroundings has been known as "chameleon" sequence. We obtained the free-energy landscape of this sequence by using a generalized-ensemble method, multicanonical molecular dynamics simulation, to sample the conformational space. The system was expressed with an all-atom model in explicit water, and the initial conformation for the simulation was a random one. The free-energy landscape demonstrated that this sequence inherently has an ability to form either alpha or beta structure: The conformational distribution in the landscape consisted of two alpha-helical clusters with different packing patterns of hydrophobic residues, and four beta-hairpin clusters with different strand-strand interaction patterns. Narrow pathways connecting the clusters were found, and analysis on the pathways showed that a compact structure formed at the N-terminal root of the chameleon sequence controls the cluster-cluster transitions. The free-energy landscape indicates that a small conditional change induces alpha-beta transitions. Additional unfolding simulations done with replacing amino acids showed that the chameleon sequence has an advantage to form an alpha-helix. Current study may be useful to understand the mechanism of diseases resulting from abnormal chain folding, such as amyloid disease. 相似文献
68.
Effect of high phosphate concentration on osteoclast differentiation as well as bone-resorbing activity 总被引:1,自引:0,他引:1
Kanatani M Sugimoto T Kano J Kanzawa M Chihara K 《Journal of cellular physiology》2003,196(1):180-189
Although high inorganic phosphate (Pi) concentration in culture media directly inhibits generation of new osteoclasts and also inhibits bone resorption by mature osteoclasts, its precise mechanism and the physiological role have not been elucidated. The present study was performed to investigate these issues. Increase in extracellular Pi concentration ([Pi](e)) (2.5-4 mM) concentration dependently inhibited 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] or parathyroid hormone (PTH)-(1-34)-induced osteoclast-like cell formation from unfractionated bone cells in the presence of stromal cells. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited 1,25(OH)(2)D(3)-, PTH-(1-34)-, or receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced osteoclast-like cell formation from hemopoietic blast cells in the absence of stromal cells. Increase in [Pi](e) (2.5-4 mM) dose dependently stimulated the expression of osteoprotegerin (OPG) mRNA and increased the expression of OPG mRNA suppressed by PTH-(1-34) or 1,25(OH)(2)D(3) in unfractionated bone cells, while it did not affect RANKL mRNA. Increase in [Pi](e) (2.5-4 mM) concentration dependently inhibited the bone-resorbing activity of isolated rabbit osteoclasts. Increase in [Pi](e) (4 mM) induced the apoptosis of isolated rabbit osteoclasts while it did not affect the apoptosis of osteoclast precursor cells and mouse macrophage-like cell line C7 cells that can differentiate into osteoclasts in the presence of RANKL and M-CSF. These results indicate that increase in [Pi](e) inhibits osteoclast differentiation both by up-regulating OPG expression and by direct action on osteoclast precursor cells. It is also indicated that increase in [Pi](e) inhibits osteoclastic activity at least in part by the direct induction of apoptosis of osteoclasts. 相似文献
69.
Homocysteine induces vascular endothelial growth factor expression in differentiated THP-1 macrophages 总被引:10,自引:0,他引:10
Hyperhomocysteinemia has been reported to be an independent risk factor for atherosclerosis and atherothrombosis. However, the molecular mechanism by which hyperhomocysteinemia can lead to atherosclerosis and atherothrombosis has not been completely described. Vascular endothelial growth factor (VEGF) has been proposed to play an important role in the progression of atherosclerosis. In the present study, we hypothesized that hyperhomocysteinemia might be associated with VEGF expression in atherosclerotic lesions. We investigated VEGF mRNA expression and VEGF secretion by homocysteine (Hcy) in differentiated THP-1 macrophages. As a result, it has been revealed that VEGF mRNA was upregulated by Hcy in a dose- and time-dependent manner in THP-1 macrophages with the increase in VEGF secretion. Importantly, other sulfur compounds, such as methionine and cysteine, showed no effect on VEGF expression, indicating that homocysteine specifically induced VEGF. Our findings suggest that hyperhomocysteinemia could promote the development of atherosclerotic lesions through VEGF induction in macrophages. 相似文献