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113.
The ability to metabolically label proteins with 35S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of 35S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression. 相似文献
114.
I. N. Semenchuk L. A. Taranova A. A. Kalenyuk P. V. Il’yasov A. N. Reshetilov 《Applied Biochemistry and Microbiology》2000,36(1):69-72
The operating and storage stability of a receptor element of an amperometric biosensor based on thePseudomonas rathonis strain T capable of degrading surfactants was tested. Microbial cells were immobilized by incorporation in gels (agar, agarose,
and calcium-alginate), polyvinyl alcohol membrane, adhesion to Chromatographic paper GF/A, or by cross-linking induced by
glutaric aldehyde. Incorporation of microbial cells in agar gel provides long-standing conservation of their activity and
viability during measurements of high concentrations of surfactants and allows the receptor element of the biosensor to be
rapidly recovered after measurements. 相似文献
115.
Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions. 相似文献
116.
117.
Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae). 总被引:4,自引:0,他引:4
A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1-11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of alpha-thrombin (K(i) = 4.89 +/- 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for gamma-thrombin (K(i) = 22.3 +/- 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin. 相似文献
118.
D S Faffe G H Silva P M Kurtz E M Negri V L Capelozzi P R Rocco W A Zin 《Journal of applied physiology》2001,90(4):1400-1406
The dynamic mechanical properties of lung tissue and its contents of collagen and elastic fibers were studied in strips prepared from mice instilled intratracheally with saline (C) or silica [15 (S15) and 30 days (S30) after instillation]. Resistance, elastance, and hysteresivity were studied during oscillations at different frequencies on S15 and S30. Elastance increased from C to silica groups but was similar between S15 and S30. Resistance was augmented from C to S15 and S30 and was greater in S30 than in S15 at higher frequencies. Hysteresivity was higher in S30 than in C and S15. Silica groups presented a greater amount of collagen than did C. Elastic fiber content increased progressively along time. This increment was related to the higher amount of oxytalan fibers at 15 and 30 days, whereas elaunin and fully developed elastic fibers were augmented only at 30 days. Silicosis led not only to pulmonary fibrosis but also to fibroelastosis, thus assigning a major role to the elastic system in the silicotic lung. 相似文献
119.
Disseminated nocardiosis in three macaque monkeys 总被引:1,自引:0,他引:1
Extrapulmonary nocardiosis was diagnosed at necropsy in two rhesus monkeys (Macaca mulatta) and one pigtailed monkey (M. nemestrina) over a four-year period in a large primate center. Typical lesions were multiple pyogranulomatous foci in the liver, intestines, peritoneum, lung and brain. Partially acid-fast, branching, filamentous organisms were seen in all lesions. Nocardia sp. was isolated from two cases. We postulate that two of the monkeys were infected by the oral route because of the distribution of lesions. 相似文献
120.
Mild chymotrypsin digestion of native lipase (449 amino acids) preferentially cleaved the Phe 335-Ala 336 bond. On SDS-gel electrophoresis, 3 major bands were observed: band 1 (52 kDa) representing native lipase, bands 2 and 3 (40 and 12 kDa) representing the two lipase fragments A and B. Fragment A does not retain lipase activity but maintains its ability to adsorb to interfaces. Fragment B was identified with the lipase C-terminal region (336-449). It does not exhibit any activity towards tributyrylglycerol emulsions and any ability to adsorb to interfaces. 相似文献