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991.
Hassan AE Shortnacy-Fowler AT Montgomery JA Secrist JA 《Nucleosides, nucleotides & nucleic acids》2000,19(3):559-565
A convenient synthesis of 2'-deoxy-2-fluoro-adenosine (1) is described. Deaminative fluorination of 2-aminoadenosine (2) followed by silylation of the 3', 5'-hydroxyl groups gave the corresponding 2-fluoroadenosine derivative 4 in good yield. Thiocarbonylation of 4 to thiocarbonylimidazolyl derivative 5a followed by treatment with an excess of tris(trimethylsilyl)silane (TTMSS) and tert-butyl peroxide in toluene at 80 degrees C was found to affect an efficient deoxygenation to the corresponding 2'-deoxy derivative 6. Desilylation of 6 by Et4NF in CH3CN afforded 1 in high yield. 相似文献
992.
Attempts were made to improve the growth of endophytic fungus Acremonium sp. and its raw sago starch degrading enzyme (RSSDE) production using different nitrogen and carbon sources at varying pH values and temperatures. It was observed that growth and enzyme activity levels were highest with peptone and sodium nitrate as the nitrogen sources and raw sago starch as the carbon source of which the optimum concentrations were 0.5 g/l, 3 g/l, and 20 g/l, respectively. Cell growth and RSSDE production reached their optimum at pH 5.0 and incubation temperature of 30 degrees C. Under these conditions, the enzyme production was significantly increased by 19- to 22-folds compared to the activity obtained in the original basal medium. 相似文献
993.
Ali YB Carrière F Verger R Petry S Muller G Abousalham A 《Journal of lipid research》2005,46(5):994-1000
Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase. 相似文献
994.
995.
Petry S Ben Ali Y Chahinian H Jordan H Kleine H Müller G Carrière F Abousalham A 《Journal of lipid research》2005,46(3):603-614
The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors. 相似文献
996.
Krimbou L Hajj Hassan H Blain S Rashid S Denis M Marcil M Genest J 《Journal of lipid research》2005,46(8):1668-1677
It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways. 相似文献
997.
In this study, engorged Amblyomma lepidum ticks were found to drop off in two peaks, one in the morning and one in the evening. Most larvae and females engorged during the morning hours between 06.00 h and 10.00 h with a peak around 08.00 h, whereas the majority of the nymphs dropped in the evening between 18.00 h and 24.00 h with the peak around 22.00 h. Although the effect of time on drop-off patterns of the ticks was statistically significant (p≤ 0.001), there were no significant seasonal influences. Survival of unfed stages of A. lepidum was also studied and was found to increase from larvae to adult ticks. The longest survival periods of 10, 11 and 14 weeks were recorded during the wet season for larvae, nymphs and adults, respectively. It is concluded that environmental conditions required for survival of A. lepidum are optimal only during the wet season and that during other seasons the tick depends primarily on prevailing micro-climatic conditions for its survival. 相似文献
998.
Genetics in the age of systems biology 总被引:3,自引:0,他引:3
Systems biology has become a fashionable label for a new generation of large-scale experiments. This essay explores how classical approaches such as forward genetics fit into this emerging framework. 相似文献
999.
1000.
Jeon WB Aceti DJ Bingman CA Vojtik FC Olson AC Ellefson JM McCombs JE Sreenath HK Blommel PG Seder KD Burns BT Geetha HV Harms AC Sabat G Sussman MR Fox BG Phillips GN 《Journal of structural and functional genomics》2005,6(2-3):143-147
The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR. 相似文献