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941.
高效的植物DNA提取方法 总被引:22,自引:0,他引:22
利用液氮研磨植物幼芽,以苯酚─氯仿─异戊醇─核糖核酸酶法提取了大豆、菜豆、玉米、高梁等几种农作物的总DNA。所提取的DNA经岛津UV-265紫外分光光度计检测及0.6%的琼脂糖凝胶电泳,结果证明:该方法所提取的植物总DNA纯度高,片段长度整齐,约50kb左右,符合外源DNA导入作物的要求,并且DNA收率很高。 相似文献
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943.
Ya-Nan Wang Wei-Hong He Hua He Xun Du Bin Jia Zhi-Peng Zeng Ming-Li An Guo-Can Chen 《Archives of microbiology》2012,194(10):809-813
A Gram-negative, non-mobile, polar single flagellum, rod-shaped bacterium WZBFD3-5A2T was isolated from a wheat soil subjected to herbicides for several years. Cells of strain WZBFD3-5A2T grow optimally on Luria-Bertani agar medium at 30?°C in the presence of 0–4.0?% (w/v) NaCl and pH 8.0. 16S rRNA gene sequence analysis revealed that strain WZBFD3-5A2T belongs to the genus Pseudomonas. Physiological and biochemical tests supported the phylogenetic affiliation. Strain WZBFD3-5A2T is closely related to Pseudomonas nitroreducens IAM1439T, sharing 99.7?% sequence similarity. DNA–DNA hybridization experiments between the two strains showed only moderate reassociation similarity (33.92?±?1.0?%). The DNA G+C content is 62.0?mol%. The predominant respiratory quinine is Q-9. The major cellular fatty acids present are C16:0 (28.55?%), C16:1ω6c or C16:1ω7c (20.94?%), C18:1ω7c (17.21?%) and C18:0 (13.73?%). The isolate is distinguishable from other related members of the genus Pseudomonas on the basis of phenotypic and biochemical characteristics. From the genotypic, chemotaxonomic and phenotypic data, it is evident that strain WZBFD3-5A2T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas nitritereducens sp. nov. is proposed. The type strain is WZBFD3-5A2T (=CGMCC 1.10702T?=?LMG 25966T). 相似文献
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945.
946.
Su H Carter CB Fröhlich O Cummings RD Chen G 《American journal of physiology. Renal physiology》2012,303(2):F201-F208
Urea transporters UT-A1 and UT-A3 are both expressed in the kidney inner medulla. However, the function of UT-A3 remains unclear. Here, we found that UT-A3, which comprises only the NH(2)-terminal half of UT-A1, has a higher urea transport activity than UT-A1 in the oocyte and that this difference was associated with differences in N-glycosylation. Heterologously expressed UT-A3 is fully glycosylated with two glycoforms of 65 and 45 kDa. By contrast, UT-A1 expressed in HEK293 cells and oocytes exhibits only a 97-kDa glycosylation form. We further found that N-glycans of UT-A3 contain a large amount of poly-N-acetyllactosamine. This highly glycosylated UT-A3 is more stable and is enriched in lipid raft domains on the cell membrane. Kifunensine, an inhibitor of α-mannosidase that inhibits N-glycan processing beyond high-mannose-type N-glycans, significantly reduced UT-A3 urea transport activity. We then examined the native UT-A1 and UT-A3 glycosylation states from kidney inner medulla and found the ratio of 65 to 45 kDa in UT-A3 is higher than that of 117 to 97 kDa in UT-A1. The highly stable expression of highly glycosylated UT-A3 on the cell membrane in kidney inner medulla suggests that UT-A3 may have an important function in urea reabsorption. 相似文献
947.
Youn JY Wang T Blair J Laude KM Oak JH McCann LA Harrison DG Cai H 《American journal of physiology. Heart and circulatory physiology》2012,302(11):H2243-H2249
The endothelial nitric oxide synthase (eNOS) requires tetrahydrobiopterin (H(4)B) as a cofactor and, in its absence, produces superoxide (O(2)(·-)) rather than nitric oxide (NO(·)), a condition referred to as eNOS uncoupling. DOCA-salt-induced hypertension is associated with H(4)B oxidation and uncoupling of eNOS. The present study investigated whether administration of sepiapterin or H(4)B recouples eNOS in DOCA-salt hypertension. Bioavailable NO(·) detected by electron spin resonance was markedly reduced in aortas of DOCA-salt hypertensive mice. Preincubation with sepiapterin (10 μmol/l for 30 min) failed to improve NO(·) bioavailability in hypertensive aortas while it augmented NO(·) production from control vessels, implicating a hypertension-associated deficiency in sepiapterin reductase (SPR), the rate-limiting enzyme for sepiapterin conversion to H(4)B. Indeed, a decreased SPR expression was observed in aortic endothelial cells, but not in endothelium-denuded aortic remains, implicating an endothelium-specific SPR deficiency. Administration of hypertensive aortas with H(4)B (10 μmol/l, 30 min) partially restored vascular NO(·) production. Combined administration of H(4)B and the NADPH oxidase inhibitor apocynin (100 μmol/l, 30 min) fully restored NO(·) bioavailability while reducing O(2)(·-) production. In angiotensin II-induced hypertension, however, aortic endothelial SPR expression was not affected. In summary, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension, due to an endothelium-specific loss in SPR, whereas coadministration of H(4)B and apocynin is highly efficient in recoupling eNOS. This is consistent with our previous observations that in angiotensin II hypertension, endothelial deficiency in dihydrofolate reductase is alternatively responsible for uncoupling of eNOS. Taken together, these data indicate that strategies specifically targeting at different H(4)B metabolic enzymes might be necessary in restoring eNOS function in different types of hypertension. 相似文献
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949.
SN Shehata RW Hunter E Ohta MW Peggie HJ Lou F Sicheri E Zeqiraj BE Turk K Sakamoto 《Cellular signalling》2012,24(11):2085-2094
PCTAIRE-1 (cyclin-dependent kinase [CDK] 16) is a highly conserved serine/threonine kinase that belongs to the CDK family of protein kinases. Little is known regarding PCTAIRE-1 regulation and function and no robust assay exists to assess PCTAIRE-1 activity mainly due to a lack of information regarding its preferred consensus motif and the lack of bona fide substrates. We used positional scanning peptide library technology and identified the substrate-specificity requirements of PCTAIRE-1 and subsequently elaborated a peptide substrate termed PCTAIRE-tide. Recombinant PCTAIRE-1 displayed vastly improved enzyme kinetics on PCTAIRE-tide compared to a widely used generic CDK substrate peptide. PCTAIRE-tide also greatly improved detection of endogenous PCTAIRE-1 activity. Similar to other CDKs, PCTAIRE-1 requires a proline residue immediately C-terminal to the phosphoacceptor site (+1) for optimal activity. PCTAIRE-1 has a unique preference for a basic residue at +4, but not at +3 position (a key characteristic for CDKs). We also demonstrate that PCTAIRE-1 binds to a novel cyclin family member, cyclin Y, which increased PCTAIRE-1 activity towards PCTAIRE-tide >100-fold. We hypothesised that cyclin Y binds and activates PCTAIRE-1 in a way similar to which cyclin A2 binds and activates CDK2. Point mutants of cyclin Y predicted to disrupt PCTAIRE-1-cyclin Y binding severely prevented complex formation and activation of PCTAIRE-1. We have identified PCTAIRE-tide as a powerful tool to study the regulation of PCTAIRE-1. Our understanding of the molecular interaction between PCTAIRE-1 and cyclin Y further facilitates future investigation of the functions of PCTAIRE-1 kinase. 相似文献
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