Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells: a method to restore cell-specific beta-trace glycoprotein productivity at high cell density.

The impact of different cultivation-infection strategies on the productivity of baculovirus-infected BTI-Tn-5B1-4 (High Five) cells was investigated. Using beta-trace protein as the recombinant glycoprotein, the effects of multiplicity of infection (MOI) and time of infection (TOI) were studied on growth after infection as well as the degree of infection and recombinant protein productivity in batch culture. The highest productivities were found when infecting Tn5 cells at early exponential growth phase (EGP) (low cell density) using a high MOI. To increase the productive cell density of Tn5 cells after beta-trace-baculovirus infection, we performed studies infecting cells in the range of 1 to 5 x 10(6) cells/mL in fresh medium. Although the protein production was increased twofold, a strong negative cell density effect was still observed when maximal productive cell density exceeded 1 x 10(6) cells/mL. To verify whether the changing cell environment of the batch experiments was responsible for the decrease in protein productivity at increasing cell density at infection, several perfusion experiments were designed by infecting Tn5 cells at cell densities over 2 x 10(6) cells/mL under more steady-state conditions. The use of this experimental setup enabled successful infections at high cell densities with volumetric productivities of up to 1.2 g L(-1) day(-1) of beta-trace protein, which is very high for a glycoprotein expressed with the baculovirus expression vector system (BEVS). The cell specific protein productivity observed after infections at higher cell densities in perfusion mode was the same as in batch experiments at low cell concentrations, which clearly demonstrates that the cell density effect could be completely overcome with perfusion cultivation.

Effect of ammonium ions on the aerobic glycolysis in Ehrlich ascites tumor cells.

The effect of ammonium ions on aerobic glycolysis has been studied in Ehrlich ascites tumor cells. The inhibition of respiration caused by 5-25 mM glucose, fructose or mannose is increased by the presence of 0.5-20 mM NH4+. Under these conditions, more glucose is used and a crossover analysis of intermediate metabolites of glycolysis indicates an increase in the glycolytic flux. In spite of the stimulation of glycolysis, the production of lactate decreases in the presence of ammonium ions, although the concentration of free alanine (glutamate and aspartate) is increased. Ammonium ions enhance the oxidation of cytochromes c and a caused by glucose, and decrease the adenine nucleotide pool and the ATP/ADP ratio. These effects of ammonium ions are observed when glucose is present in the incubation medium, and do not occur when only ammonium salts were supplied to the cells metabolizing endogenous substrates.

Ammonia overloading in hepatocytes isolated from liver of fetal and adult rats.

Ammonia overloading was investigated during glucose and fructose metabolism in isolated hepatocytes under a variety of metabolic conditions. In all assay conditions, the glycolytic flux and oxygen uptake was not modified by 10 mM ammonia. In hepatocytes isolated from rats fed as libitum, the presence of ammonia caused a decrease in the production of lactate (pyruvate); this effect was not observed in anaerobic incubations, in hepatocytes isolated from starved animals, or in fetal hepatocytes. In spite of an overproduction of urea, ammonia detoxification also takes place by the synthesis of alanine, glutamate and aspartate. Addition of 1 mM aminooxyacetate, an inhibitor of aminotransferases, to the incubation medium prevents the formation of these amino acids, and also prevents the decrease of lactate in hepatocytes isolated from fed animals.

Crabtree effect induced by fructose in hepatocytes isolated from developing rats.

The inhibition of respiration caused by fructose is investigated in hepatocytes isolated from fetal and developing rats. The respiration rate of the hepatocytes increases in the early days after birth. Adult values for oxygen uptake are reached by hepatocytes isolated from 14-day-old rats. After weaning, the Crabtree effect induced by fructose appears simultaneously with changes in energy metabolism.

L-Alanine as an end product of glycolysis in Saccharomyces cerevisiae growing under different hypoxic conditions.

Saccharomyces cerevisiae growing under anaerobic or other hypoxic conditions releases L-alanine into the culture medium as an end product of glycolysis. Although the production of alanine is not as high as that of other fermentation products (ethanol, glycerol, succinic acid), consideration of the pathways leading to alanine in fermenting yeasts indicates that the release of alanine is advantageous to the cellular economy and may be considered as a safety device for excreting reducing equivalents derived from NADPH. No significant changes in the activity of alanine aminotransferase are found in the yeast when grown under different conditions.