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排序方式: 共有262条查询结果,搜索用时 15 毫秒
1.
A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing. 总被引:6,自引:4,他引:2 下载免费PDF全文
A simple and rapid procedure for the preparation of M13 single stranded DNA sequencing templates which does not involve phenol extractions and alcohol precipitations is described. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. Subsequent dissociation of the phages and removal of contaminants is performed while the DNA is bound to the glass. Finally, the purified DNA is eluted in a small volume of low-salt buffer. The yield is higher than that obtained by standard methods. The simplified procedure takes less than 30 minutes and does not demand special skills or equipment; the sequence resolution is as good as that obtained by standard procedures both with the Klenow fragment and T7 DNA polymerase, with radioactive labelling as well as in automated sequencing with a fluorescent label. 相似文献
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An antibody against secretogranin I (chromogranin B) is packaged into secretory granules 总被引:16,自引:8,他引:8 下载免费PDF全文
P Rosa U Weiss R Pepperkok W Ansorge C Niehrs E H Stelzer W B Huttner 《The Journal of cell biology》1989,109(1):17-34
We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles. 相似文献
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Assumpció Bosch Stefan Wiemann Jordi Guimerá Wilhelm Ansorge David Patterson Xavier Estivill 《Human genetics》1995,95(1):119-122
Five clones, containing polymorphic CA-repeat sequences, have been isolated from a specific human chromosome 21 phage library and have been localised to band q21 of chromosome 21 using a somatic cell hybrid panel. These highly repetitive sequences (D21S1263, D21S1264, D21S1415, D21S1417 and D21S1420) have been characterised in the CEPH reference parents and have heterozygosities ranging from 0.30 to 0.81 and an average polymorphism information content (PIC) of 0.62. The relative order of these markers, based on the somatic cell hybrid panel, is cen-D21S1417, D21S1420-D21S1263, D21S1415-D21S1264-tel. The most polymorphic marker (D21S1264) has been included in the chromosome 21 genetic map. They have also been localised in the CEPH/ Généthon YAC panel, providing a refined localisation of these polymorphic sequences. These five CA-repeat markers should provide a better characterisation of the q21 region of chromosome 21. 相似文献
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B Wiederanders S Ansorge P Bohley U Broghammer H Kirschke J Langner 《Acta biologica et medica Germanica》1976,35(3-4):269-283
The preparation and properties of cathepsin D from rat liver are reported. The enzyme is an endopeptidase of lysosomal origin. The molecular weight was estimated to be 49000 by sodium-dodecylsulfate electrophoresis. We did not find any dissociation into subunits under reducing conditions, in contrast to some other authors. We found the enzyme to occur in at least 4 forms with the isoelectric points 5.87, 5.65, 5.41 and 5.13. Strong -SH-blocking reagents inhibit the activity, but the most powerful and specific inhibitor was pepstatin (Ki=38 nM). The substrate specificity is discussed. There was no proof for any zymogen activation in a great number of experiments. Since the cathepsins B1, B3 and L obviously seem to play the major role in the intracellular protein breakdown within the rat liver, the main task of cathepsin D is the degradation of extracellular proteins in this organ. 相似文献
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Alessio Atzori Viveka N. Malviya Giuliano Malloci Jürg Dreier Klaas M. Pos Attilio V. Vargiu Paolo Ruggerone 《生物化学与生物物理学报:生物膜》2019,1861(1):62-74
Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-OprM, the major efflux pump transporter in Pseudomonas aeruginosa. However, not much is known regarding recognition and transport of these compounds by AcrAB-TolC, which is the MexAB-OprM homologue in Escherichia coli and by definition the paradigm model for structural studies on efflux pumps. Prompted by this motivation, we unveiled the molecular details of the interaction of imipenem and meropenem with the transporter AcrB by combining computer simulations with biophysical experiments. Regarding the interaction with the two main substrate binding regions of AcrB, the so-called access and deep binding pockets, molecular dynamics simulations revealed imipenem to be more mobile than meropenem in the former, while comparable mobilities were observed in the latter. This result is in line with isothermal titration calorimetry, differential scanning experiments, and binding free energy calculations, indicating a higher affinity for meropenem than imipenem at the deep binding pocket, while both sharing similar affinities at the access pocket. Our findings rationalize how different physico-chemical properties of compounds reflect on their interactions with AcrB. As such, they constitute precious information to be exploited for the rational design of antibiotics able to evade efflux pumps. 相似文献
9.
Dolby V Lundqvist A Fröberg T Lüllau E Shaw J Tjerneld F Cronet P 《Journal of biochemical and biophysical methods》2004,58(3):195-205
The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, d-Phe7]-alphaMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 degrees C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor. 相似文献
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