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1.
Impaired secretion and fluid-phase endocytosis in the End4 mutant of Chinese hamster ovary cells 总被引:6,自引:0,他引:6
R H Wang P A Colbaugh C Y Kao E A Rutledge R K Draper 《The Journal of biological chemistry》1990,265(33):20179-20187
Mutant V.24.1 defines the End4 complementation group of temperature-sensitive Chinese hamster ovary cell mutants selected for resistance to protein toxins. We investigated the secretory pathway in the mutant cells and found: 1) The hemagglutinin of influenza virus failed to reach the plasma membrane and was retained in a form sensitive to endoglycosidase H at the restrictive temperature. 2) Transferrin receptors synthesized at the restrictive temperature remained sensitive to endoglycosidase H. 3) Secretion of total soluble protein into the medium was strongly reduced at high temperature. These data indicate that V.24.1 cells are defective in secretion at the restrictive temperature. To see what effect the lesion had on the endocytic pathway, we measured the accumulation and recycling of the fluid-phase marker horseradish peroxidase. Accumulation was inhibited by 50% while recycling was barely affected, suggesting that the rate of fluid-phase endocytosis was reduced. We previously showed that the clathrin-coated pit pathway of endocytosis was not affected in the mutant, indicated by a normal transferrin cycle (Colbaugh, P. A., Stookey, M., and Draper, R. K. (1989) J. Cell Biol. 108, 2211-2219). Thus, the secretory lesion correlates with reduced fluid-phase endocytosis without impairing the clathrin-dependent pathway of receptor-mediated endocytosis. We also investigated the delivery of endocytosed material to lysosomes and found that delivery was partially, but not completely, impaired in the mutant. This suggests that endocytosed material can enter lysosomes, although slowly, in the absence of a functional secretory pathway. 相似文献
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4.
Draper J.; Davey M. R.; Freeman J. P.; Cocking E. C.; Cox B. J. 《Plant & cell physiology》1982,23(3):451-458
Suspension cell protoplasts of albino Petunia hybrida have beentransformed by isolated Agrobacterium tumefaciens Ti plasmid.Uptake of octopine Ti plasmid (pTiACH5) into protoplasts wasstimulated by poly-L-ornithine and polyethylene glycol (PEG).The frequency and efficiency of transformation of protoplaststo phytohormone autotrophy was compared using the two uptakeagents with various concentrations of plasmid. Transformationwas most efficient with PEG-mediated uptake, 5 µg of Tiplasmid per 106 protoplasts giving a frequency of 6?105.Octopine was not synthesised in any of the transformants afterthe second subculture on hormone-free medium. DNA-DNA hybridisationshowed the presence of DNA homologous to the T-DNA region ofpTiACH5 in all clones analysed. (Received November 9, 1981; Accepted January 29, 1982) 相似文献
5.
Characterization of a transferrin-diphtheria toxin conjugate 总被引:3,自引:0,他引:3
We report here the synthesis and properties of a hybrid toxin prepared by covalently coupling diphtheria toxin to transferrin. The purified material contained two major hybrid protein species and was highly cytotoxic to mouse LMTK- cells in culture, reducing protein synthesis by 50% in 24 h at a concentration of 1 ng/ml. Cytotoxic activity was completely abolished in the presence of exogenous transferrin or anti-transferrin or anti-diphtheria toxin, thus demonstrating that the hybrid toxin was intoxicating cells via their transferrin receptors and that both the diphtheria toxin and transferrin components of the conjugate were necessary for activity. NH4Cl, a drug that elevates the pH within acidic intracellular vesicles, also blocked cytotoxic activity, suggesting that a low intravesicular pH was required for activity. The inhibitory effect of NH4Cl could be abolished by exposing toxin-treated cells to acidic culture medium, further implicating an acid-dependent step in the mechanism of the hybrid toxin action. Studies on the kinetics of intoxication also implied that endocytosis and exposure to a low pH within vesicles were necessary for cytotoxicity. Altogether, the results suggest that the transferrin-diphtheria toxin conjugate binds to transferrin receptors and is internalized into acidic endocytic vesicles. The enzymatic moiety of diphtheria toxin then apparently enters the cytosol in response to the low pH and subsequently arrests protein synthesis. 相似文献
6.
Anorexia Nervosa and Respecting a refusal of life-prolonging Therapy: A Limited Justification 总被引:1,自引:0,他引:1
Heather Draper 《Bioethics》2000,14(2):120-133
People who suffer from eating disorders often have to be treated against their will, perhaps by being detained, perhaps by being forced to eat. In this paper it is argued that whilst forcing compliance is generally acceptable, there may be circumstances under which a sufferer's refusal of consent to treatment should be respected. This argument will hinge upon whether someone in the grip of an eating disorder can actually make competent decisions about their quality of life. If so, then the decision to refuse therapy may be on a par with other decisions to refuse life-prolonging therapy made by sufferers of debilitating chronic, or acute onset terminal illness. In such cases, palliation might justifiably replace aggressive therapy. The argument will also draw heavily on the distinction between competent refusal of therapy and passive euthanasia, and the distinction between incompetent and irrational decisions. Both distinctions will then be applied to decisions to refuse food. The extent to which sufferers from anorexia nervosa can be categorised as either incompetent or irrational will be examined. It is against this background that it will be argued that at least some of those who suffer from eating disorders should have their refusals respected, even if they may die as a result. 相似文献
7.
The protein quality of barley was determined by the measurement of the protein efficiency ratio (PER). Available lysine in the same samples was determined chemically and by microbiological assay, and relationships were obtained between PER and either total or available lysine content.In the PER test, groundnut meal was found to be a suitable protein supplement to barley for the purpose of detecting differences in the protein quality of barley. The method was sufficiently sensitive to detect a reduction in protein quality of one variety as a result of micronization. Available lysine values obtained by the modified Tetrahymena method were significantly correlated (r = 0.99) with PER values; there was no significant correlation between total lysine content and the PER value.The available lysine values measured by the two chemical methods were higher than the Tetrahymena values. The Silcock method gave results better correlated (r = 0.93) with the Tetrahymena values than did the Carpenter method (r = 0.82). The results of chemical methods were not significantly related with PER values, but the methods ranked the barleys in an order similar to that obtained with the PER and Tetrahymena tests. 相似文献
8.
A potentiometric study of the flavin semiquinone equilibrium 总被引:3,自引:0,他引:3
9.
K G Draper 《Biochemical and biophysical research communications》1989,163(1):466-470
An analysis of the human albumin mRNA structure revealed a nonrandom distribution of self-complementary regions within the mRNA. The majority of these self-complementary mRNA stretches encode important structural regions of the human albumin protein. The amino acids contained within these regions of the protein exhibit a high degree of hydrophobic complementarity which could influence local protein conformation and contribute to the biological importance of the protein structures. 相似文献
10.
N-2-(Propenal) serine (S-MDA) was synthesized by reacting serine with malondialdehyde (MDA) and was shown to be a 1:1 adduct of the starting materials. The synthetic compound was found to be identical to a metabolite of MDA excreted in rat and human urine. The identity of the metabolite was confirmed by isolation and hydrolysis to yield equimolar quantities of serine and MDA. The presence of S-MDA in urine constitutes direct evidence for oxidative decomposition of phospholipids by lipid peroxidation in vivo. 相似文献