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排序方式: 共有641条查询结果,搜索用时 15 毫秒
71.
72.
Laetitia Malphettes Yevgeniy Freyvert Jennifer Chang Pei‐Qi Liu Edmond Chan Jeffrey C. Miller Zhe Zhou Thu Nguyen Christina Tsai Andrew W. Snowden Trevor N. Collingwood Philip D. Gregory Gregory J. Cost 《Biotechnology and bioengineering》2010,106(5):774-783
IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8?/? cell lines was fourfold higher than in the best‐producing clones of FUT8?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc. 相似文献
73.
74.
Mazères G Leloup L Daury L Cottin P Brustis JJ 《Cell motility and the cytoskeleton》2006,63(4):193-207
The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced mu- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed. 相似文献
75.
Saidi H Eslahpazir J Eslaphazir J Carbonneil C Carthagena L Requena M Nassreddine N Belec L 《Journal of immunology (Baltimore, Md. : 1950)》2006,177(8):5540-5549
Human lactoferrin (Lf) is an iron binding glycoprotein that is present in several mucosal secretions. Many biological functions have been ascribed to Lf. In the present study, we showed that Lf limited specifically adsorption of R5- and X4-HIV-1-free particles on endometrial epithelial HEC-1A cells, by inhibiting virus adsorption on heparan-sulfated proteoglycans. But, Lf did not interfere with both R5 and X4-HIV transcytosis. We showed also the efficacy of Lf in preventing R5 and X4-HIV capture by dendritic cells. Conversely, we demonstrated that Lf-reacting natural Abs (NAbs) present within i.v. Ig-enhanced HIV attachment on dendritic cells by forming HIV-Lf-NAbs. HIV particles were able to directly interact with Lf following its interaction with NAbs. We also found Lf-reacting natural Abs within cervicovaginal secretions, suggesting the existence of Lf-NAbs complexes in women genital tract in vivo. In conclusion, this study highlights Lf as a potent microbicides and reports new function for NAbs within the genital compartment that may compartment that may abolish the inhibitory activity of microbicide compounds. Thus, we proposed a model in which Lf would appear as a double-edged sword that could have beneficial or detrimental effects depending on both cellular and molecular environments. This study highlights the use of Lf derivates as microbicide candidates to limit such interferences. 相似文献
76.
The structures of crustacean hyperglycemic hormones (CHH) were investigated in two crabs, the coastal euryhaline crab Pachygrapsus marmoratus and the fresh water crab Potamon ibericum. The neuropeptide mRNAs were extracted from pericardial and X-organs (PO and XO), and the sequences of the cDNA encoding the hormones' precursors were determined. The X-organ preprohormones are composed of 29 and 28 amino acid signal peptides in P. marmoratus and P. ibericum respectively, followed by 43 and 41 amino acid crustacean hyperglycemic hormone precursor related peptide (CPRP) flanking the 72 amino acid crustacean hyperglycemic hormones. A similar organization is reported for pericardial preprohormones with identical sequences for the signal peptide, the CPRP and the N-terminal sequences of CHH (1-40), but remaining sequences (41-72 and 41-71) differing considerably. In P. marmoratus two CHH cDNAs were characterized from XO and evidences were obtained for the existence of at least two forms in the PO. From our results and by comparison with other known sequences, a consensus pattern for crab pericardial CHH could be pointed out. Analysis of the data presented in this article using phylogenetic methods reveals that the two crab species studied are much closer than previously predicted. 相似文献
77.
78.
Lanzer M Wickert H Krohne G Vincensini L Braun Breton C 《International journal for parasitology》2006,36(1):23-36
Discovered in 1902 by Georg Maurer as a peculiar dotted staining pattern observable by light microscopy in the cytoplasm of erythrocytes infected with the human malarial parasite Plasmodium falciparum, the function of Maurer's clefts have remained obscure for more than a century. The growing interest in protein sorting and trafficking processes in malarial parasites has recently aroused the Maurer's clefts from their deep slumber. Mounting evidence suggests that Maurer's clefts are a secretory organelle, which the parasite establishes within its host erythrocyte, but outside its own confines, to route parasite proteins across the host cell cytoplasm to the erythrocyte surface where they play a role in nutrient uptake and immune evasion processes. Moreover, Maurer's clefts seem to play a role in cell signaling, merozoite egress, phospholipid biosynthesis and, possibly, other biochemical pathways. Here, we review our current knowledge of the ultrastructure of Maurer's clefts, their proteinaceous composition and their function in protein trafficking. 相似文献
79.
High pressure-low temperature processing of food proteins 总被引:1,自引:0,他引:1
High pressure-low temperature (HP-LT) processing is of interest in the food field in view of: (i) obtaining a "cold" pasteurisation effect, the level of microbial inactivation being higher after pressurisation at low or sub-zero than at ambient temperature; (ii) limiting the negative impact of atmospheric pressure freezing on food structures. The specific effects of freezing by fast pressure release on the formation of ice I crystals have been investigated on oil in water emulsions stabilized by proteins, and protein gels, showing the formation of a high number of small ice nuclei compared to the long needle-shaped crystals obtained by conventional freezing at 0.1 MPa. It was therefore of interest to study the effects of HP-LT processing on unfolding or dissociation/aggregation phenomena in food proteins, in view of minimizing or controlling structural changes and aggregation reactions, and/or of improving protein functional properties. In the present studies, the effects of HP-LT have been investigated on protein models such as (i) beta-lactoglobulin, i.e., a whey protein with a well known 3-D structure, and (ii) casein micelles, i.e., the main milk protein components, the supramolecular structure of which is not fully elucidated. The effects of HP-LT processing was studied up to 300 MPa at low or sub-zero temperatures and after pressure release, or up to 200 MPa by UV spectroscopy under pressure, allowing to follow reversible structural changes. Pressurisation of approximately 2% beta-lactoglobulin solutions up to 300 MPa at low/subzero temperatures minimizes aggregation reactions, as measured after pressure release. In parallel, such low temperature treatments enhanced the size reduction of casein micelles. 相似文献
80.
Séron K Corset L Vasseur F Boutin P Gómez-Ambrosi J Salvador J Frühbeck G Froguel P 《Biochemical and biophysical research communications》2006,348(4):1232-1238
Animal studies have illustrated the importance of the expression in adipose tissue of the leptin receptor (OB-R), and of SOCS3 an inhibitor of the leptin signaling pathway, in body weight regulation. The aim of the present study was to investigate in human adipose tissues of the same patients the OB-R isoforms and SOCS3 expression. Subcutaneous and omental adipose tissues were obtained from 6 lean and 18 morbidly obese women. The long isoform OB-Rb mRNA mediating leptin signaling, and SOCS3 mRNA are abundantly present in the subcutaneous fat of lean women, but are 90% and 70% decreased (P<0.0001) in obese women. In visceral fat from lean and obese women, both OB-Rb and SOCS3 mRNA are detected at very low levels. Subcutaneous/visceral ratios for OB-Ra the short OB-R isoform, OB-Rb, and SOCS3 mRNA abundance strongly correlate with the insulin sensitivity index, HOMA-% S, (r=0.49, P<0.0001, r=0.42, P=0.0002 and r=0.38, P=0.0002, respectively) in both lean and obese patients without type 2 diabetes. The near absence of OB-Rb mRNA and the similarly decreased SOCS3 expression in obese adipose tissue may reflect a defective leptin signaling pathway that could play a role in the impairment of insulin sensitivity associated with excess adiposity. 相似文献