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1.
The increasing accessibility of mass isotopomer data via GC-MS and NMR technology has necessitated the use of a systematic and reliable method to take advantage of such data for flux analysis. Here we applied a nonlinear, optimization-based method to study substrate metabolism in cardiomyocytes using (13)C data from perfused mouse hearts. The myocardial metabolic network used in this study accounts for 257 reactions and 240 metabolites, which are further compartmentalized into extracellular space, cytosol, and mitochondrial matrix. Analysis of the perfused mouse heart showed that the steady-state ATP production rate was 16.6 +/- 2.3 micromol/min . gww, with 30% of the ATP coming from glycolysis. Of the four substrates available in the perfusate (glucose, pyruvate, lactate, and oleate), exogenous glucose forms the majority of cytosolic pyruvate. Pyruvate decaboxylation is significantly higher than carboxylation, suggesting that anaplerosis is low in the perfused heart. Exchange fluxes were predicted to be high for reversible enzymes in the citric acid cycle (CAC), but low in the glycolytic pathway. Pseudoketogenesis amounted to approximately 50% of the net ketone body uptake. Sensitivity analysis showed that the estimated flux distributions were relatively insensitive to experimental errors. The application of isotopomer data drastically improved the estimation of reaction fluxes compared to results computed with respect to reaction stoichiometry alone. Further study of 12 commonly used (13)C glucose mixtures showed that the mixtures of 20% [U-(13)C(6)] glucose, 80% [3 (13)C] glucose and 20% [U-(13)C(6)] glucose, 80% [4 (13)C] were best for resolving fluxes in the current network. 相似文献
2.
The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC8 (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC5 in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC8. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC5 expansion has now been extended to LTC-IC8 expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation. 相似文献
3.
Jan Schellenberger Junyoung O Park Tom M Conrad Bernhard Ø Palsson 《BMC bioinformatics》2010,11(1):213
Background
Genome-scale metabolic reconstructions under the Constraint Based Reconstruction and Analysis (COBRA) framework are valuable tools for analyzing the metabolic capabilities of organisms and interpreting experimental data. As the number of such reconstructions and analysis methods increases, there is a greater need for data uniformity and ease of distribution and use. 相似文献4.
5.
Craig R. Halberstadt Bernhard O. Palsson A. Rees Midgley Rane L. Curl 《Biotechnology and Bioprocess Engineering》2002,7(3):163-170
This report describes the use of a transtubular bioreactor to study the relative effects of diffusion versus perfusion of
medium on antibody production by a hybridoma cell line. The study was performed with a high-density cell culture maintained
in a serum-free, low-protein medium for 77 days. It was determined that the reactor possessed a macro-mixing pattern residence
time distribution similar to a continuous stirred tank reactor (CSTR). However, due to the arrangement of the medium lines
in the reactor, the flow patterns for nutrient distribution consist of largely independent medium path lengths ranging from
short to long. When operated with cyclic, reversing, transtubular medium flow, some regions of the reactor (with short residence
times) are more accessible to medium than others (with long residence times). From this standpoint, the reactor can be divided
into three regions: a captive volume, which consists of medium primarily delivered via diffusion; a lapped volume, which provides
nutrients through unilateral convection; and a swept volume, which operates through bilateral convection. The relative sizes
of these three volumes were modified experimentally by changing the period over which the direction of medium flow was reversed
from 15 min (larger captive volume) to 9 h (larger swept volume). The results suggest that antibody concentration increases
as the size of the diffusion-limited (captive) volume is increased to a maximum at around 30 min with a sharp decrease thereafter.
As reflected by changes in measured consumption of glucose and production of lactate, no significant difference in cellular
metabolism occurred as the reactor was moved between these different states. These results indicate that the mode of operation
of the transtubular bioreactor may influence antibody productivity under serum-free, low-protein conditions with minimal effects
on cellular metabolism. 相似文献
6.
The structure of calcitonin isolated from salmon1 (SCT 1) has been recently established2 and confirmed by synthesis8. It possesses an exceptionally high level of activity, exhibiting about 20–50 times the hypocalcaemic potency of any other calcitonin isolated from mammalian species. 相似文献
7.
8.
9.
The rhodopsin system of the squid 总被引:6,自引:19,他引:6
Squid rhodopsin (λmax 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λmax 500 mµ) near neutrality, yellow (λmax 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H+ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions. 相似文献
10.
Fong SS Burgard AP Herring CD Knight EM Blattner FR Maranas CD Palsson BO 《Biotechnology and bioengineering》2005,91(5):643-648
The development and validation of new methods to help direct rational strain design for metabolite overproduction remains an important problem in metabolic engineering. Here we show that computationally predicted E. coli strain designs, calculated from a genome-scale metabolic model, can lead to successful production strains and that adaptive evolution of the engineered strains can lead to improved production capabilities. Three strain designs for lactate production were implemented yielding a total of 11 evolved production strains that were used to demonstrate the utility of this integrated approach. Strains grown on 2 g/L glucose at 37 degrees C showed lactate titers ranging from 0.87 to 1.75 g/L and secretion rates that were directly coupled to growth rates. 相似文献