Effects of NHE1 expression level on CHO cell responses to environmental stress

Ammonia, lactate and CO(2) inhibit animal cell growth. Accumulation of these metabolic byproducts also causes a decrease in intracellular pH (pH(i)). Transport systems regulate pH(i) in eukaryotic cells. Ion transporters have been cloned and overexpressed in cells but have not been examined for protection against the buildup of ammonia, lactate or CO(2). The Na(+)/H(+) exchangers (NHE) transport H(+) ions from cells during acidification to increase pH(i). We examined whether overexpression of NHE1 would provide CHO cells with greater protection from elevated ammonia, lactate or CO(2). NHE1 CHO cells were compared to MT2-1-8 ("normal" levels of NHE) and AP-1 (devoid of any NHE activity) CHO cell lines. Expression of at least "normal" levels of NHE1 is necessary for CHO cell survival during exposure to 30 mM lactic acid without pH adjustment or to 20 mM NH(4)Cl with pH adjustment. Resistance to an acute acid-load increased when NHE1 was overexpressed in CHO cells. Surprisingly, the inhibitory effect on cell growth at 195 mmHg pCO(2)/435 mOsm/kg (normal levels are 40 mmHg pCO(2)/ 320 mOsm/kg) was not affected by the NHE1 level. Also, there was no further decrease in CHO cell growth in the absence of NHE1 expression during elevated osmolality alone (up to 575 mOsm/kg).     

Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study

Partial pressure of CO2 (pCO2) and osmolality as high as 150 mmHg and 440 mOsm/kg, respectively, were observed in large-scale CHO cell culture producing an antibody-fusion protein, B1. pCO2 and osmolality, when elevated to high levels in bioreactors, can adversely affect cell culture and recombinant protein production. To understand the sole impact of pCO2 or osmolality on CHO cell growth, experiments were performed in bench-scale bioreactors allowing one variable to change while controlling the other. Elevating pCO2 from 50 to 150 mmHg under controlled osmolality (about 350 mOsm/kg) resulted in a 9% reduction in specific cell growth rate. In contrast, increasing osmolality resulted in a linear reduction in specific cell growth rate (0.008 h(-1)/100 mOsm/kg) and led to a 60% decrease at 450 mOsm/kg as compared to the control at 316 mOsm/kg. This osmolality shift from 316 to 445 mOsm/kg resulted in an increase in specific production rates of lactate and ammonia by 43% and 48%, respectively. To elucidate the effect of high osmolality and/or pCO2 on the production phase, experiments were conducted in bench-scale bioreactors to more closely reflect the pCO2 and osmolality levels observed at large scale. Increasing osmolality to 400-450 mOsm/kg did not result in an obvious change in viable cell density and product titer. However, a further increase in osmolality to 460-500 mOsm/kg led to a 5% reduction in viable cell density and a 8% decrease in cell viability as compared to the control. Final titer was not affected as a result of an apparent increase in specific production rate under this increased osmolality. Furthermore, the combined effects from high pCO2 (140-160 mmHg) and osmolality (400-450 mOsm/kg) caused a 20% drop in viable cell density, a more prominent decrease as compared to elevated osmolality alone. Results obtained here illustrate the sole effect of high pCO2 (or osmolality) on CHO cell growth and demonstrate a distinct impact of high osmolality and/or pCO2 on production phase as compared to that on growth phase. These results are useful to understand the response of the CHO cells to elevated pCO2 (and/or osmolality) at a different stage of cultivation in bioreactors and thus are valuable in guiding bioreactor optimization toward improving protein production.     

Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.     

Effects of CO2 and osmolality on hybridoma cells: growth, metabolism and monoclonal antibody production

CO2 partial pressure (pCO2) in industrial cell culture reactors may reach 150–200 mm Hg, which can significantly inhibit cell growth and recombinant protein production. Due to equilibrium with bicarbonate, increased pCO2 at constant pH results in a proportional increase in osmolality. Hybridoma AB2-143.2 cell growth rate decreased with increasing pCO2 in well-plate culture, with a 45% decrease at 195 mm Hg with partial osmolality compensation (to 361 mOsm kg- 1). Inhibition was more extensive without osmolality compensation, with a 63% decrease in growth rate at 195 mm Hg and 415 mOsm kg-1. Also, the hybridoma death rate increased with increasing pCO2, with 31- and 64-fold increases at 250 mm Hg pCO2 for 401 and 469 mOsm kg- 1, respectively. The specific glucose consumption and lactate production rates were 40–50% lower at 140 mm Hg pCO2. However, there was little further inhibition of glycolysis at higher pCO2. The specific antibody production rate was not significantly affected by pCO2 or osmolality within the range tested. Hybridomas were also exposed to elevated pCO2 in continuous culture. The viable cell density decreased by 25–40% at 140 mm Hg. In contrast to the well-plate cultures, the death rate was lower at the new steady state at 140 mm Hg. This was probably due to higher residual nutrient and lower byproduct levels at the lower cell density (at the same dilution rate), and was associated with increased cell-specific glucose and oxygen uptake. Thus, the apparent effects of pCO2 may vary with the culture system. VMdZ and RK contributed equally to the results in this article. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of elevated dissolved carbon dioxide concentrations on growth of Corynebacterium glutamicum on D-glucose and L-lactate

The effect of increased dissolved carbon dioxide concentrations on growth of Corynebacterium glutamicum was studied with continuous turbidostatic cultures. The carbon sources were either l-lactate or d-glucose. To increase the dissolved carbon dioxide concentration the carbon dioxide partial pressure of the inlet gas stream pCO2,IN was increased stepwise from 0.0003 bar (air) up to 0.79 bar, while the oxygen partial pressure of the inlet gas stream was kept constant at 0.21 bar. For each resulting carbon dioxide partial pressure pCO2 the maximum specific growth rate mu(max) was determined from the feed rate resulting from the turbidostatic control. On d-glucose and pCO2 up to 0.26 bar, mu(max) was mostly constant around 0.58 h(-1). Higher pCO2 led to a slight decrease of mu(max). On l-lactate mu(max) increased gradually with increasing carbon dioxide partial pressures from 0.37 h(-1) under aeration with air to a maximum value of 0.47 h(-1) at a pCO2 of 0.26 bar. At very high pCO2 (0.81 bar) mu(max) decreased down to 0.35 h(-1) independent of the carbon source.     

Decreased pCO(2) accumulation by eliminating bicarbonate addition to high cell-density cultures

High-density perfusion cultivation of mammalian cells can result in elevated bioreactor CO(2) partial pressure (pCO(2)), a condition that can negatively influence growth, metabolism, productivity, and protein glycosylation. For BHK cells in a perfusion culture at 20 x 10(6) cells/mL, the bioreactor pCO(2) exceeded 225 mm Hg with approximate contributions of 25% from cellular respiration, 35% from medium NaHCO(3), and 40% from NaHCO(3) added for pH control. Recognizing the limitations to the practicality of gas sparging for CO(2) removal in perfusion systems, a strategy based on CO(2) reduction at the source was investigated. The NaHCO(3) in the medium was replaced with a MOPS-Histidine buffer, while Na(2)CO(3) replaced NaHCO(3) for pH control. These changes resulted in 63-70% pCO(2) reductions in multiple 15 L perfusion bioreactors, and were reproducible at the manufacturing-scale. Bioreactor pCO(2) values after these modifications were in the 68-85 mm Hg range, pCO(2) reductions consistent with those theoretically expected. Low bioreactor pCO(2) was accompanied by both 68-123% increased growth rates and 58-92% increased specific productivity. Bioreactor pCO(2) reduction and the resulting positive implications for cell growth and productivity were brought about by process changes that were readily implemented and robust. This philosophy of pCO(2) reduction at the source through medium and base modification should be readily applicable to large-scale fed-batch cultivation of mammalian cells.     

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 muM Cu, 80 muM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 muM, falling sharply at 20 muM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. (c) 1992 John Wiley & Sons, Inc.     

Hyperosmotic stress and elevated pCO2 alter monoclonal antibody charge distribution and monosaccharide content

Medium osmolality increases with pCO2 at constant pH. Elevated pCO2 and osmolality inhibit hybridoma growth to similar extents in both serum-containing and serum-free media. The combination of osmolality and elevated pCO2 synergizes to negatively impact cell growth. IgG2a glycosylation by hybridoma cells was evaluated under elevated pCO2 (to 250 mmHg pCO2) and/or osmolality (to 476 mOsm/kg). IgG2a site occupancy did not change significantly under any of the conditions studied, which is consistent with the robust glycosylation of other antibodies produced under various environmental stresses. However, changes were observed in the IgG2a charge distribution. Changes in the isoelectric point (pI) were greater under hyperosmotic stress, increasing by 0.32 and 0.41 pH units at 435 mOsm/kg in serum-containing and serum-free medium, respectively. Hyperosmotic stress also resulted in a concomitant increase in the heterogeneity of the charge distribution. The mean pI in serum-containing medium decreased by 0.16 pH units at 250 mmHg pCO2 when osmolality was controlled at 320 mOsm/kg but increased by 0.20 pH units when the osmolality increased with pCO2 (195 mmHg pCO2-435 mOsm/kg). In serum-free medium, elevated pCO2 did not alter pI, regardless of medium osmolality. In contrast to elevated osmolality at control pCO2, elevated pCO2 did not significantly alter the IgG2a charge heterogeneity under any of the conditions studied. The IgG2a was not sialylated, so sialylation changes were not responsible for changes in the charge distribution. IgG2a galactose content decreased with elevated osmolality, as a result of either elevated NaHCO3 or NaCl. However, when osmolality was controlled at elevated pCO2, the galactose content tended to increase. The mannose content decreased with increasing stress, while the fucose content remained relatively unchanged. It is likely that the observed increases in the pI of murine IgG2a were due to increased organellar pH, which is reflected by increased specific beta-galactosidase activity in the supernatant.     

Enhancement of erythropoietin production in recombinant Chinese hamster ovary cells by sodium lactate addition

The stabilization of optimum pH for cells can cause a higher erythropoietin (EPO) production rate and a good growth rate with the prolonged culture span in recombinant Chinese hamster ovary (r-CHO) cells. Our strategy for stabilizing the optimum pH in this study is to reduce the lactate production by adding sodium lactate to a culture medium. When 40 mM sodium lactate was added, a specific growth rate was decreased by approximately 22% as compared with the control culture. However the culture longevity was extended to 187 h, and more than a 2.7-fold increase in a final accumulated EPO concentration was obtained at 40 mM of sodium lactate. On the condition that caused the high production of EPO, a specific glucose consumption rate and lactate production rate decreased by 23.3 and 52%, respectively. Activity of lactate dehydrogenase (LDH) in r-CHO cells increased and catalyzed the oxidation of lactate to pyruvate, together with the reverse reaction, at the addition of 40 mM sodium lactate. The addition of 40 mM sodium lactate caused the positive effects on a cell growth and an EPO production in the absence of carbon dioxide gas as well as in the presence of carbon dioxide gas by reducing the accumulation of lactate.     

Cultivation of hybridoma cells in continuous cultures: kinetics of growth and product formation

Mouse hybridoma cells were grown in suspension in continuous stirred bioreactors. Cell growth, substrate utilization, and monoclonal antibody (MAb) production were studied using serum-free medium. Steady-state data were obtained at different dilution rates, between 0.012 and 0.039 h(-1) Viability was profoundly affected by dilution rate, particularly near the lower end of the dilution-rate range investigated. MAb concentration and productivity went through a maximum with respect to dilution rate. Lactate yield on glucose declined with in creasing dilution rate. Experiments were carried out to study the effects of medium glucose concentration on cell growth, product formation, and lactate yield on glucose. Reduction of glucose concentration in the feed medium did not considerably affect cell density and MAb concentration in the culture, but lactate levels dropped sharply; lactate yield on glucose declined substantially, indicating alterations in cell metabolic path ways for energy metabolism. Optimization strategy for continuous cell culture is discussed.     

Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality

Elevated pCO(2) inhibits cell growth. This growth inhibition is accompanied by a decrease in intracellular pH (pHi), as well as a decrease in glycolysis. Elevated concentrations (mM) of some amino acids have been shown by others to protect cells exposed to two very different environmental stresses: nutrient starvation and hyperosmolality. The fact that many of the amino acids shown to have protective effects against other stresses are transported into the cell through a pHi-sensitive transporter led us to study the possibility of using these amino acids as protective agents under elevated pCO(2). Screening experiments using 5, 15, and 25 mM of each amino acid showed that not all amino acids that protect cells from hyperosmolality protect them from elevated pCO(2). Glycine betaine and glycine were chosen for further characterization in both hybridoma and CHO cells. Asparagine and threonine were also tested in hybridoma and CHO cells, respectively. All amino acids tested under 195 mm Hg pCO(2)/435 mOsm/kg (50% growth inhibition) restored the specific growth rate (mu) in hybridoma cells to that observed under control conditions (40 mm Hg/320 mOsm/kg). Addition of each amino acid resulted in an increase in the consumption rate and intracellular accumulation of that amino acid. In CHO cells, glycine betaine also restored mu to control values, while glycine and threonine partially restored mu. In hybridoma cells, the higher specific antibody productivity obtained at elevated pCO(2) was maintained with the lowest amino acid concentration (5 mM). Productivity decreased toward control values with increasing amino acid concentrations. Elevated pCO(2) decreased the specific tPA productivity in the CHO cell line studied. Only glycine betaine resulted in a 20% increase in productivity at 195 mm Hg/435 mOsm/kg. With the exception of glycine betaine in hybridoma cells, amino acids did not mitigate the associated pHi decrease of at least 0.2 pH units at 195 mm Hg/435 mOsm/kg. pHi in hybridoma cells under elevated pCO(2) in the presence of glycine betaine was about 0.1 pH units below that of control. Amino acids had no effect on the cell size response of hybridoma cells, while they partially offset the increase in CHO cell size at elevated pCO(2). Glycine betaine, asparagine, and glycine increased the specific glucose consumption rate observed at 195 mm Hg/435 mOsm/kg (50% of control) to values greater than 70% of control in hybridoma cells. In CHO cells, only glycine betaine increased q(glc) (by 20%) under elevated pCO(2). All amino acids tested improved the cell yield from glutamine at 195 mm Hg/435 mOsm/kg in both cell lines.     

Fed-batch cultivation of the docosahexaenoic-acid-producing marine alga Crypthecodinium cohnii on ethanol

The heterotrophic marine microalga Crypthecodinium cohnii produces docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications. So far, DHA production has been studied with glucose and acetic acid as carbon sources. This study investigates the potential of ethanol as an alternative carbon source for DHA production by C. cohnii. In shake-flask cultures, the alga was able to grow on ethanol. The specific growth rate was optimal with 5 g l(-1) ethanol and growth did not occur at 0 g l(-1) and above 15 g l(-1). By contrast, in fed-batch cultivations with a controlled feed of pure ethanol, cumulative ethanol addition could be much higher than 15 g l(-1), thus enabling a high final cell density and DHA production. In a representative fed-batch cultivation of C. cohnii with pure ethanol as feed, 83 g dry biomass l(-1), 35 g total lipid l(-1) and 11.7 g DHA l(-1) were produced in 220 h. The overall volumetric productivity of DHA was 53 mg l(-1 )h(-1), which is the highest value reported so far for this alga.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Influence of CO(2)-HCO(3) Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO(2)-HCO(3) growth conditions and suggest that there exists a threshold level of CO(2) for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO(2)-HCO(3) growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K') was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO(2)-HCO(3) as a consequence of regulating electron sink metabolism.     

Optimization of substrate feed for continuous production of lactic acid by Lactococcus lactis IO-1

Summary Optimization of substrate feed for continuous production of lactic acid by the homofermentative bacterium, Lactococcus lactis IO-1, in glucose medium was investigated. A pH-dependent feed with two pH set-points, a lower set-point for neutralization with alkali and an upper set-point for substrate feed, proved better than continuous substrate feed with one pH set-point for neutralization with alkali only. Built-in electrodialysis with a cell-recycling system was tested and high cell density was achieved as a result of the use of enriched medium. However, specific lactate productivity in this system was not satisfactorily high. pH-dependent feed was combined with turbidity control and a cell recycling. With this system, we achieved high specific lactate productivity of 2 g (g-cell)^-1 h^-1 at a dilution rate of 0.5 h^-1, a dry cell weight of 5 g l ^-1, a level of lactate in the broth of 20 g l ^-1, and a concentration of glucose in the spent medium of about 5 gl ^-1.     

Effects of phentolamine on hepatic PO2 in endotoxemia

The effect of phentolamine, an alpha-adrenergic blocker, on hepatic oxygen supply, plasma glucose, and lactate, and survival in fasted male rats administered Echerichia coli endotoxin (25 mg/kg, ip) has been studied. Survival at 24 h was 8% in untreated endotoxic rats, 83% in rats receiving phentolamine (5 mg/kg, ip) and endotoxin, and 100% in phentolamine controls. Measurements during the initial 8 h postendotoxin recorded transiently lower systemic arterial pressure in the phentolamine-endotoxic rats. Arterial PO2 and increases of pH and heart rate were similar in both endotoxic groups. Lactacidemia, present by 4 h in untreated endotoxic rats, did not develop in the phentolamine group and plasma glucose was significantly higher at 8 h (98 +/- 2.5 vs. 77 +/- 5.6 mg%, mean +/- SE). Mean hepatic PO2 at 6 h in phentolamine-endotoxic rats was 9.6 mmHg with 28% of the values below 5 mmHg. By contrast, the mean in untreated endotoxic rats was 1.9 mmHg with 88% of values below 5 mmHg. Phentolamine controls were stable over 8 h; mean hepatic PO2 was 17.7 mmHg. The differences in plasma glucose and lactate suggest protection of hepatic metabolism in phentolamine-treated endotoxic rats by prevention of excessive hepatic hypoxia.     

Glucose metabolism by lymphocytes, macrophages, and tumor cells from Walker 256 tumor-bearing rats supplemented with fish oil for one generation

Here we investigated the effect of lifelong supplementation of the diet with coconut fat (CO, rich in saturated fatty acids) or fish oil (FO, rich in n-3 polyunsaturated fatty acids) on tumor growth and lactate production from glucose in Walker 256 tumor cells, peritoneal macrophages, spleen, and gut-associated lymphocytes. Female Wistar rats were supplemented with CO or FO prior to mating and then throughout pregnancy and gestation and then the male offspring were supplemented from weaning until 90 days of age. Then they were inoculated subcutaneously with Walker 256 tumor cells. Tumor weight at 14 days in control rats (those fed standard chow) and CO supplemented was approximately 30 g. Supplementation of the diet with FO significantly reduced tumor growth by 76%. Lactate production (nmol h(-1) mg(-1) protein) from glucose by Walker 256 cells in the group fed regular chow (W) was 381.8 +/- 14.9. Supplementation with coconut fat (WCO) caused a significant reduction in lactate production by 1.6-fold and with fish oil (WFO) by 3.8-fold. Spleen lymphocytes obtained from W and WCO groups had markedly increased lactate production (553 +/- 70 and 635 +/- 150) when compared to non-tumor-bearing rats ( approximately 260 +/- 30). FO supplementation reduced significantly the lactate production (297 +/- 50). Gut-associated lymphocytes obtained from W and WCO groups increased lactate production markedly (280 +/- 31 and 276 +/- 25) when compared to non-tumor-bearing rats ( approximately 90 +/- 18). FO supplementation reduced significantly the lactate production (168 +/- 14). Lactate production by peritoneal macrophages was increased by tumor burden but there was no difference between the groups fed the various diets. Lifelong consumption of FO protects against tumor growth and modifies glucose metabolism in Walker tumor cells and lymphocytes but not in macrophages.     

L-valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum

Corynebacterium glutamicum was engineered for the production of L-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum DeltaaceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, L-alanine, and L-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum DeltaaceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and L-alanine towards L-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum DeltaaceE(pJC4ilvBNCE) produced up to 210 mM L-valine with a volumetric productivity of 10.0 mM h(-1) (1.17 g l(-1) h(-1)) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose.     

Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells

The aim of the present work was to study the effect of ammonia and lactate on growth, metabolism, and productivity of BHK cells producing a recombinant fusion protein. Results show that cell growth was reduced with the increase in ammonia or lactate: k(1/2) of 1.1 mM and 3.5 mM for stirred and stationary cultures, respectively, for ammonia and of 28 mM for both stationary and stirred cultures for lactate, were obtained. The cell-specific consumption rates of both glucose (q(Glc)) and glutamine (q(Gln)) increased, whereas that of oxygen (q(O2)) decreased, with the increase in ammonia or lactate concentrations. The cell-specific production rates of lactate (q(Lac)) increased with an increase in ammonia concentration; similarly for the cell-specific production rates of ammonia (q(Amm)), which also increased with an increase in lactate concentration; on the other hand, both q(Lac) and q(Amm) markedly decreased when lactate or ammonia concentrations were increased, respectively; lactate was consumed at lactate concentrations above 30 mM and ammonia was consumed at ammonia concentrations above 5 mM. In vivo (31)P NMR experiments showed that ammonia and lactate affect the intracellular pH, leading to intracellular acidification, and decrease the content in phosphomonoesters, whereas the cell energy state was maintained. The effect of lactate on cell growth and q(Gln) is partially due to osmolarity, on q(Glc) and q(Amm) is entirely due to osmolarity, but on q(Lac) is mainly due to lactate effect per se. An increase in ammonia from 0 to 20 mM induced a 50% reduction in specific productivity, whereas an increase in lactate from 0 to 60 mM induced a 40% decrease.     

Alanine Uptake and Release by Sympathetic Ganglia of Chicken Embryos

Uptake and release of alanine were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14-15 days old, and incubated in a modified Eagle's minimum essential medium. In the presence of [U-14C]glucose, glucose carbon accumulated in alanine in the medium at a rate that increased when unlabeled alanine was added and sometimes exceeded the rate of appearance in lactate. When combined with uptake data, the increase in appearance of labeled alanine in the medium could be accounted for quantitatively by interference with its reuptake, without assuming a change in the unidirectional output of labeled alanine, provided allowance was made for the measured properties of exchange between the extracellular space and the surrounding medium. According to this model, the constant unidirectional outflux of labeled alanine was about 50 mumol/g dry weight/h. When [U-14C]alanine was added to medium containing unlabeled glucose, the alanine was consumed at a rate that increased as the concentration of alanine in the medium was elevated. The uptake rate was found to fit a modified Michaelis-Menten equation with a Umax of about 120 mumol/g dry weight/h, a Km of 0.5-1.0 mM, and a Kd of 0.75 ml/g dry weight/h. By chemical measurement of changes in alanine concentration in the medium during incubation, the uptake rate was shown to equal the output rate when about 0.2 mM alanine was present. Much of the alanine consumed in the presence of glucose was metabolized to CO2, raising the total CO2 output above the rate obtained with glucose alone. When alanine was present at a concentration of 10-20 mM, it contributed almost as much carbon to CO2 as did the glucose. A higher percentage of the carbon from alanine was incorporated into tissue constituents than was carbon from either glucose or lactate. It is concluded that alanine can be significant both as a product and as a substrate, but that its role as substrate would not be great at typical concentrations of alanine in blood.