Activity of glutamate dehydrogenase is increased in ammonia-stressed hybridoma cells

The effect of added ammonia on the intracellular fluxes in hybridoma cells was investigated by metabolic-flux balancing techniques. It was found that, in ammonia-stressed hybridoma cells, the glutamate-dehydrogenase flux is in the reverse direction compared to control cells. This demonstrates that hybridoma cells are able to prevent the accumulation of ammonia by converting ammonia and alpha-ketoglutarate into glutamate. The additional glutamate that is produced by this flux, as compared to the control culture, is converted by the reactions catalyzed by alanine aminotransferase (45% of the extra glutamate) and aspartate aminotransferase (37%), and a small amount is used for the biosynthesis of proline (6%). The remaining 12% of the extra glutamate is secreted into the culture medium. The data suggest that glutamate dehydrogenase is a potential target for metabolic engineering to prevent ammonia accumulation in high-cell-density culture.     

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.     

Metabolic flux analysis of hybridoma cells in different culture media using mass balances

The estimation of the intracellular fluxes of mammalian cells using only the mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. Either additional experimental flux data or additional theoretical constraints are required to find one unique flux distribution out of the solution space that is bound by the mass balances. Here, a method is developed using the latter approach. The uptake and production rates of amino acids, glucose, lactate, O(2), CO(2), NH(4), MAB, and the intracellular amino acid pools have been determined for two different steady-states. The cellular composition {total protein and protein composition, total lipids and fatty acid distribution, total carbohydrates, DNA and RNA} has been measured to calculate the requirements for biosynthesis. It is shown to be essential to determine the uptake/production rates of ammonia and either carbon dioxide or oxygen. In mammalian cells these are cometabolites of cyclic metabolic pathways. The flux distribution that is found using the Euclidean minimum norm as the additional theoretical constraint and taking either the CO(2) or the NAD(P)H mass balance into account is shown to be in agreement with the measured O(2) and CO(2) metabolic rates.The metabolic fluxes in hybridoma cells in continuous culture at a specific growth rate of 0.83 day(-1) are estimated for a medium with (optimal medium) and without (suboptimal medium) Primatone RL, an enzymatic hydrolysate of animal tissue that causes a more than twofold increase in cell density. It is concluded that (i)The majority of the consumed glucose (>90%) is channeled through the pentose-phosphate pathway in rapidly proliferating cells.(ii)Pyruvate oxidation and tricarboxylic acid (TCA) cycle activity are relatively low, i.e., 8% of the glucose uptake in suboptimal and 14% in optimal medium, respectively. Under both conditions, only a small fraction of pyruvate is further oxidized to CO(2).(iii)The flux from glutamate to alpha-ketoglutarate (catalyzed by glutamate dehydrogenase) is almost zero in medium with and even slightly reversed in medium without Primatone RL. Almost all glutamate enters the TCA cycle due to the action of transaminases.(iv)Transhydrogenation plays a significant role in hybridoma cells under our experimental conditions. NADPH is produced at relatively high rates (11 x 10(-12) to 13 x 10(-12) mol . cell(-1) . day(-1)) compared to other fluxes in both culture media. (c) 1996 John Wiley & Sons, Inc.     

Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l⁻¹. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l⁻¹. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l⁻¹ has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l⁻¹ h⁻¹. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb⁻¹ by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291. Received 12 September 2000/ Accepted in revised form 27 January 2001     

The effect of diethyl- -fluoroglutarate on phosphates and glutamate in slices of rabbit cerebral cortex

Abstract— The diethyl ester of α-fluoroglutarate (DEFG), an inhibitor of glutamate dehydrogenase, was prepared, and its effect on glutamate and phosphates in slices of rabbit cerebral cortex was examined. The primary effect of the drug on cortical slices incubating in a Krebs-Ringer glucose medium was to decrease the tissue levels of glutamate in association with decreased levels and turnover of high-energy phosphates. Assimilation of exogenous glutamate by the slices was partially blocked in the presence of the drug and severely depressed oxidative phosphorylation resulted when glutamate and DEFG were both present in the incubation mixture. The results suggested a significant relationship between the activity of cerebral glutamate dehydrogenase and oxidative phosphorylation. During incubation in a Krebs-Ringer glucose medium the endogenous pool of free amino acids in the cortical slice partitioned with the medium. Little or no glutamate, aspartate or GABA was present in the medium after incubation, but glycine, alanine, threonine, serine and glutamine did partition to varying degrees, with over one-half of the glutamine present in the incubation medium. With the exception of ‘leakage’ of aspartate, the partitioning patterns were relatively unaffected by the presence of added glutamate or DEFG.     

An overview of the serpin superfamily

Serpins are a broadly distributed family of protease inhibitors that use a conformational change to inhibit target enzymes. They are central in controlling many important proteolytic cascades, including the mammalian coagulation pathways. Serpins are conformationally labile and many of the disease-linked mutations of serpins result in misfolding or in pathogenic, inactive polymers.