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Inhibition kinetics of phenol degradation from unstable steady-state data   总被引:4,自引:0,他引:4  
Multiplicity of steady states of a continuous culture with an inhibitory substrate was used to estimate kinetic parameters under steady-state conditions. A continuous culture of Pseudomonas cepacia G4, using phenol as the sole source of carbon and energy, was overloaded by increasing the dilution rate above the critical dilution rate. The culture was then stabilized in the inhibitory branch by a proportional controller using the carbon dioxide concentration in the reactor exhaust gas as the controlled variable and the dilution rate as the manipulated variable. By variation of the set point, several unstable steady states in the inhibitory branch were investigated and the specific phenol conversion rates calculated. In addition, phenol degradation was investigated under substrate limitation (chemostat operation).The results show that the phenol degradation by P. cepacia can be described by the same set of inhibition parameters under substrate limitation and under high substrate concentrations in the inhibitory branch. Biomass yield and maintenance coefficients were identical. Fitting of the data to various inhibition models resulted in the best fit for the Yano and Koga equation. The well-known Haldane model, which is most often used to describe substrate inhibition by phenol, gave the poorest fit. The described method allows a precise data estimation under steady-state conditions from the maximum of the biological reaction rate up to high substrate concentrations in the inhibitory branch. Inhibition parameter estimation by controlling unstable steady states may thus be useful in avoiding discrepancies between data generated by batch runs and their application to continuous cultures which have been often described in the literature. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 567-576, 1997.  相似文献
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IRE1 and PERK are type I transmembrane serine/threonine protein kinases that are activated by unfolded proteins in the endoplasmic reticulum (ER) to signal adaptive responses. IRE1 is present in all eukaryotic cells and signals the unfolded protein response through its kinase and endoribonuclease activities. PERK signals phosphorylation of a translation initiation factor to inhibit protein synthesis in higher eukaryotic cells but is absent in the Saccharomyces cerevisiae genome. The amino acid sequences of the amino-terminal ER luminal domains (NLDs) from IRE1 and PERK display limited homology and have diverged among species. In this study, we have demonstrated that the NLD of yeast Ire1p is required for signaling. However, the NLDs from human IRE1alpha and murine IRE1beta and the Caenorhabditis elegans IRE1 and PERK function as replacements for the S. cerevisiae Ire1p-NLD to signal the unfolded protein response. Replacement of the Ire1p-NLD with a functional leucine zipper dimerization motif yielded a constitutively active kinase that surprisingly was further activated by ER stress. These results demonstrate that ER stress-induced dimerization of the NLD is sufficient for IRE1 and PERK activation and is conserved through evolution. We propose that ligand-independent activation of IRE1 and PERK permits homodimerization upon accumulation of unfolded proteins in the lumen of the ER.  相似文献
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Overexpression of recombinant proteins in animal cells is commonly achieved by using gene amplification techniques. Gene amplified cells possess up to several thousand genes coding for the target protein. Constitutive expression of these genes leads to high levels of the corresponding mRNA species and the immature protein in the cell. Inefficient processing of these precursors may result from their great abundance in the cell. To study the influence of elevated intracellular levels of a recombinant protein on its maturation and secretion, we examined the maturation and secretion of human antithrombin III (hATIII) in Chinese hamster ovary (CHO) cells at different levels of gene amplification. No loss of vitality was caused by elevated secretion of hATIII. As the intracellular hATIII content increased, the efficiency of hATIII secretion decreased steadily. The state of intracellular hATIII from the different cell lines was studied by determining the specific heparin cofactor activity of hATIII. Intracellular hATIII from the highest amplified cell line displayed a lowered specific heparin cofactor activity indicating the presence of malfolded, only partially folded, or incompletely or incorrectly posttranslationally modified hATIII in this cell line. Thus, the ability of CHO cells to fold and/or introduce posttranslational modifications and subsequently to secrete the recombinant protein becomes saturated, and therefore these processes may become limiting for protein secretion at highly elevated expression levels. This limitation was not due to a general exhaustion of the secretory capacity of the cells because hATIII constituted only a minor fraction of the secreted proteins, even at high expression levels. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 547-559, 1997.  相似文献
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Studies of rotifer community composition and dynamics often rely on limited sampling regimes. To determine how well species richness is reflected in these studies, we examined interannual variation of rotifer species richness and monogonont community structure from 10 aquatic systems comprising four habitat types—springs, rock pools (tinajas), former cattle tanks, and artificial ponds—in Big Bend National Park (Texas, USA). Planktonic, littoral, and benthic samples were collected from all sites at about the same date for each of five summers (2001–2005). Our survey yielded 15 monogonont families including 30 genera and 84 species. Two bdelloid taxa also were designated. Species richness varied widely among these four habitats: range, 1–32; mean (±1 SD), 11.2 ± 8.0. Total Species richness in the habitats also varied considerably: springs (54 taxa) > artificial ponds (35 taxa) > tinajas (19 taxa) > cattle tanks (15 taxa). Sessile species comprised ≈13% of the taxa in our samples. Species turnover indices (STI) of these systems indicate low overall relatedness: mean (±1 S.D.) = 85.2 ± 7.1%. The relative frequency of encounter of most taxa in the four systems was low, with 79 taxa (≈92%) having values ≤2.0%. Singleton rates were quite high, ranging from 46.7 to 71.4%, with an overall mean ≈65.1%. Most importantly, we found that both species richness and STI varied considerably among habitat type. Species richness varied by 2–10× between consecutive years and STI ranged from 64 to 89% over the entire study. Our results indicate that rotifer community composition fluctuates greatly over time, and that rotifer community structure may be more labile than is generally believed. Species richness and thus biodiversity may be dramatically underestimated using single sampling or short-term strategies that are often employed in studies of zooplankton community structure. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont & R. Rico-Martínez Advances in Rotifer Research  相似文献
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