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91.
92.
93.
W. F. LOOMIS C. KLEIN PH. BRACKET 《Differentiation; research in biological diversity》1979,12(1-3):83-89
Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10−5 M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+ .
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation. 相似文献
Treatment with EDTA and Mg
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation. 相似文献
94.
Immunological changes associated with age contribute to the high rates of influenza virus morbidity and mortality in the elderly. Compounding this problem, aged individuals do not respond to vaccination as well as younger, healthy adults. Efforts to increase protection to this demographic group are of utmost importance, as the proportion of the population above the age of 65 is projected to increase in the coming decade. Using a live influenza virus with a truncated nonstructural protein 1 (NS1), we are able to stimulate cellular and humoral immune responses of aged mice comparable to levels seen in young mice. Impressively, a single vaccination provided protection following stringent lethal challenge in aged mice. 相似文献
95.
96.
Krammer F Pontiller J Tauer C Palmberger D Maccani A Baumann M Grabherr R 《PloS one》2010,5(10):e13265
Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells. 相似文献
97.
Gruber K Gartler G Krammer B Schwab H Kratky C 《The Journal of biological chemistry》2004,279(19):20501-20510
The hydroxynitrile lyases (HNLs) from Hevea brasiliensis (HbHNL) and from Manihot esculenta (MeHNL) are both members of the alpha/beta-hydrolase superfamily. Mechanistic proposals have been put forward in the past for both enzymes; they differed with respect to the role of the active-site lysine residue for which a catalytic function was claimed for the Hevea enzyme but denied for the Manihot enzyme. We applied a freeze-quench method to prepare crystals of the complex of HbHNL with the biological substrate acetone cyanohydrin and determined its three-dimensional structure. Site-directed mutagenesis was used to prepare the mutant K236L, which is inactive although its three-dimensional structure is similar to the wild-type enzyme. However, the structure of the K236L-acetone cyanohydrin complex shows the substrate in a different orientation from the wild-type complex. Finite difference Poisson-Boltzmann calculations show that in the absence of Lys(236) the catalytic base His(235) would be protonated at neutral pH. All of this suggests that Lys(236) is instrumental for catalysis in several ways, i.e. by correctly positioning the substrate, by stabilizing the negatively charged reaction product CN(-), and by modulating the basicity of the catalytic base. These data complete the elucidation of the reaction mechanism of alpha/beta-hydrolase HNLs, in which the catalytic triad acts as a general base rather than as a nucleophile; proton abstraction from the substrate is performed by the serine, and reprotonation of the product cyanide is performed by the histidine residues. Together with a threonine side chain, the active-site serine and lysine are also involved in substrate binding. 相似文献
98.
99.
Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications 总被引:1,自引:0,他引:1
Evidence of associations between free-living amoebas and human disease has
been increasing in recent years. Knowledge about phylogenetic relationships
that may be important for the understanding of pathogenicity in the genera
involved is very limited at present. Consequently, we have begun to study
these relationships and report here on the phylogeny of Hartmannella
vermiformis, a free-living amoeba that can harbor the etiologic agent of
Legionnaires' disease. Our analysis is based on studies of small-subunit
ribosomal RNA genes (srDNA). Nucleotide sequences were determined for
nuclear srDNA from three strains of H. vermiformis isolated from the United
Kingdom, Germany, and the United States. These sequences then were compared
with a sequence previously obtained for a North American isolate by J. H.
Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an
average GC content of 49.6%. Sequence differences among the strains range
are 0.38%-0.76%. Variation occurs at 19 positions and includes 2
single-base indels plus 14 monotypic and 3 ditypic single-base
substitutions. Variation is limited to eight helix/loop structures
according to a current model for srRNA secondary structure. Parsimony,
distance, and bootstrap analyses used to examine phylogenetic relationships
between the srDNA sequences of H. vermiformis and other eukaryotes
indicated that Hartmannella sequences were most closely related to those of
Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent
with a separation between European and North American strains of
Hartmannella, but results of other tests of this relationship were
statistically inconclusive.
相似文献
100.
Bentele M Lavrik I Ulrich M Stösser S Heermann DW Kalthoff H Krammer PH Eils R 《The Journal of cell biology》2004,166(6):839-851
Mathematical modeling is required for understanding the complex behavior of large signal transduction networks. Previous attempts to model signal transduction pathways were often limited to small systems or based on qualitative data only. Here, we developed a mathematical modeling framework for understanding the complex signaling behavior of CD95(APO-1/Fas)-mediated apoptosis. Defects in the regulation of apoptosis result in serious diseases such as cancer, autoimmunity, and neurodegeneration. During the last decade many of the molecular mechanisms of apoptosis signaling have been examined and elucidated. A systemic understanding of apoptosis is, however, still missing. To address the complexity of apoptotic signaling we subdivided this system into subsystems of different information qualities. A new approach for sensitivity analysis within the mathematical model was key for the identification of critical system parameters and two essential system properties: modularity and robustness. Our model describes the regulation of apoptosis on a systems level and resolves the important question of a threshold mechanism for the regulation of apoptosis. 相似文献