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81.
The DNA binding domain of estrogen receptor alpha is required for high-affinity nuclear interaction induced by estradiol 总被引:1,自引:0,他引:1
Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER. 相似文献
82.
Effect of intratumoral injection of carboplatin combined with pluronic P85 or L61 on experimental colorectal carcinoma in rats 总被引:1,自引:0,他引:1
Krupka TM Weinberg BD Wu H Ziats NP Exner AA 《Experimental biology and medicine (Maywood, N.J.)》2007,232(7):950-957
Pluronic, a poly(ethylene oxide)-poly(propylene oxide)-poly (ethylene oxide) block copolymer, has been shown to enhance the cytotoxic activity of anticancer drugs in various cell lines. In the current study the effect of Pluronic P85 (P85) and Pluronic L61 (L61) on the intratumoral chemotherapy of an experimental adenocarcinoma in rats was examined. A total of 120 subcutaneous tumors (4 per rat) were inoculated in 30 BDIX rats and were treated weekly for 4 weeks with intratumoral injection of carboplatin (CPt) alone or with either P85 or L61. Tumors were monitored weekly and were excised at the endpoint for histologic evaluation. The effect of Pluronic on levels of intracellular ATP was explored as a possible mechanism of sensitization. Results showed that tumors treated with low-dose CPt (2.8 mg/kg) and P85 or L61 exhibited significant reductions in tumor volume after 28 days relative to Day 0 (112.7% +/- 34.4%, n = 15; 131.3% +/- 55.6%, n = 8) compared with tumors treated with free drug (339.4% +/- 75.0%, n = 16). Control tumors treated with either P85 or L61 alone or with saline showed volume increases of 1079.4% +/- 143.6% (n = 16), 729.4% +/- 202.2% (n = 7), and 1119.2% +/- 6.1% (n = 16), respectively. Treatment with high-dose CPt (20.7 mg/kg) led to a 79.3% +/- 4.2% reduction in tumor volume, and no differences were noted with addition of P85 or L61. In vitro ATP measurements showed that 28.0 mg/kg of P85 significantly reduced levels of intracellular ATP to 44.7% +/- 1.5% of controls, whereas L61 at this concentration depleted ATP levels completely. Results confirm that Pluronic P85 and L61 act as potent sensitizers to carboplatin chemotherapy of the experimental colorectal carcinoma, leading to a significant reduction of tumor growth compared to carboplatin alone. ATP depletion is a possible mechanism for these observed differences. 相似文献
83.
Hong Q Bakshi RK Palucki BL Park MK Ye Z He S Pollard PG Sebhat IK Liu J Guo L Cashen DE Martin WJ Weinberg DH MacNeil T Tang R Tamvakopoulos C Peng Q Miller RR Stearns RA Chen HY Chen AS Strack AM Fong TM MacIntyre DE Wyvratt MJ Nargund RP 《Bioorganic & medicinal chemistry letters》2011,21(8):2330-2334
We report the discovery of piperazine urea based compound 1, a potent, selective, orally bioavailable melanocortin subtype-4 receptor partial agonist. Compound 1 shows anti-obesity efficacy without potentiating erectile activity in the rodent models. 相似文献
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Burridge PW Thompson S Millrod MA Weinberg S Yuan X Peters A Mahairaki V Koliatsos VE Tung L Zambidis ET 《PloS one》2011,6(4):e18293
Background
The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods.Methodology/Principal Findings
We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.Conclusion/Significance
This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine. 相似文献88.
89.
Garelli FM Espinosa MO Weinberg D Trinelli MA Gürtler RE 《PLoS neglected tropical diseases》2011,5(3):e991
Background
A five-year citywide control program based on regular application of temephos significantly reduced Aedes aegypti larval indices but failed to maintain them below target levels in Clorinda, northern Argentina. Incomplete surveillance coverage and reduced residuality of temephos were held as the main putative causes limiting effectiveness of control actions.Methodology
The duration of temephos residual effects in household-owned water-holding tanks (the most productive container type and main target for control) was estimated prospectively in two trials. Temephos was applied using spoons or inside perforated small zip-lock bags. Water samples from the study tanks (including positive and negative controls) were collected weekly and subjected to larval mortality bioassays. Water turnover was estimated quantitatively by adding sodium chloride to the study tanks and measuring its dilution 48 hs later.Principal Findings
The median duration of residual effects of temephos applied using spoons (2.4 weeks) was significantly lower than with zip-lock bags (3.4 weeks), and widely heterogeneous between tanks. Generalized estimating equations models showed that bioassay larval mortality was strongly affected by water type and type of temephos application depending on water type. Water type and water turnover were highly significantly associated. Tanks filled with piped water had high turnover rates and short-lasting residual effects, whereas tanks filled with rain water showed the opposite pattern. On average, larval infestations reappeared nine weeks post-treatment and seven weeks after estimated loss of residuality.Conclusions
Temephos residuality in the field was much shorter and more variable than expected. The main factor limiting temephos residuality was fast water turnover, caused by householders'' practice of refilling tanks overnight to counteract the intermittence of the local water supply. Limited field residuality of temephos accounts in part for the inability of the larval control program to further reduce infestation levels with a treatment cycle period of 3 or 4 months. 相似文献90.
Yohannes E Ghosh SK Jiang B McCormick TS Weinberg A Hill E Faddoul F Chance MR 《PloS one》2011,6(11):e27816
The oral epithelium, the most abundant structural tissue lining the oral mucosa, is an important line of defense against infectious microorganisms. HIV infected subjects on highly active antiretroviral therapy (HAART) are susceptible to comorbid viral, bacterial and fungal infections in the oral cavity. To provide an assessment of the molecular alterations of oral epithelia potentially associated with susceptibility to comorbid infections in such subjects, we performed various proteomic studies on over twenty HIV infected and healthy subjects. In a discovery phase two Dimensional Difference Gel Electrophoresis (2-D DIGE) analyses of human oral gingival epithelial cell (HOEC) lysates were carried out; this identified 61 differentially expressed proteins between HIV-infected on HAART subjects and healthy controls. Down regulated proteins in HIV-infected subjects include proteins associated with maintenance of protein folding and pro- and anti-inflammatory responses (e.g., heat-shock proteins, Cryab, Calr, IL-1RA, and Galectin-3-binding protein) as well as proteins involved in redox homeostasis and detoxification (e.g., Gstp1, Prdx1, and Ero1). Up regulated proteins include: protein disulfide isomerases, proteins whose expression is negatively regulated by Hsp90 (e.g., Ndrg1), and proteins that maintain cellular integrity (e.g., Vimentin). In a verification phase, proteins identified in the protein profiling experiments and those inferred from Ingenuity Pathway Analysis were analyzed using Western blotting analysis on separate HOEC lysate samples, confirming many of the discovery findings. Additionally in HIV-infected patient samples Heat Shock Factor 1 is down regulated, which explains the reduced heat shock responses, while activation of the MAPK signal transduction cascade is observed. Overall, HAART therapy provides an incomplete immune recovery of the oral epithelial cells of the oral cavity for HIV-infected subjects, and the toxic side effects of HAART and/or HIV chronicity silence expression of multiple proteins that in healthy subjects function to provide robust innate immune responses and combat cellular stress. 相似文献