Comparison of a production process in a membrane-aerated stirred tank and up to 1000-L airlift bioreactors using BHK-21 cells and chemically defined protein-free medium

The applicability of a protein-free medium for the production of recombinant human interleukin-2 with baby hamster kidney cells in airlift bioreactors was investigated. For this purpose, a BHK-21 cell line, adapted to grow and produce in protein-free SMIF7 medium without forming spheroids in membrane-aerated bubble-free bioreactors, was used as the producer cell line. First, cultivation of the cells was established at a 20-L scale using an internal loop airlift bioreactor system. During the culturing process the medium formulation was optimized according to the specific requirements associated with cultivation of mammalian cells under protein-free conditions in a bubble-aerated system. The effects of the addition of an antifoam agent on growth, viability, productivity, metabolic rates, and release of lactate dehydrogenase were investigated. Although it was possible to establish cultivation and production at a 20-L scale without the use of antifoaming substances, the addition of 0.002% silicon-oil-based antifoaming reagent improved the cultivation system by completely preventing foam formation. This reduced the release of lactate dehydrogenase activity to the level found in bubble-free aerated stirred tank membrane bioreactors and led to a reduction in generation doubling times by about 5 h (17%). Using the optimized medium formulation, cells were cultivated at a 1000-L scale, resulting in a culture performance comparable to the 20-L airlift bioreactor. For comparison, cultivations with protein-containing SMIF7 medium were carried out at 20- and 1000-L scales. The application of protein supplements did not lead to a significant improvement in the cultivation conditions. The results were also compared with experiments performed in a bubble-free aerated stirred tank membrane bioreactor to evaluate the influence of bubbles on the investigated culture parameters. The data implied a higher metabolic activity of the cells in airlift bioreactors with a 150% higher glucose consumption rate. The results of this study clearly demonstrate the applicability of a protein-free chemically defined medium for the production of recombinant proteins with BHK cells in airlift bioreactors.     

Brownian diffusion and electrophoretic transport of double‐stranded DNA in agarose gels

The field free diffusion constant and the electric field dependence of the electrophoretic mobility and molecular orientation of DNA samples from 5 to 164 kilobase pairs in agarose gels from 0.5 to 2% have been measured by fluorescence recovery after photobleaching and birefringence. In conditions where the reptation predictions hold for the field free diffusion, they partially fail for the DNA size dependence of the low field limit of the electrophoretic mobility. The linear field dependencies of the electrophoretic mobility and orientation factor seem to favor the biased reptation model with fluctuations over the standard biased reptation model, which predicts a quadratic field dependence. The quantitative analysis of the molecular parameters shows, however, that most experiments have been carried out at values of the field where the difference between the two models may be less conclusive. The pore size dependence of the different quantities has been given a particular attention and the role of the distribution of pore sizes in the departures from the reptation predictions is discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 45–59, 1999     

An evolutionary perspective on the patterning of maternal investment in pregnancy

Pregnancy is thought to be a metabolically very expensive endeavor, yet investigations have produced inconsistent results concerning the responsiveness of human birth weight to maternal nutritional stress or nutritional intervention. These findings have led some researchers to conclude that fetal growth is strongly buffered against fluctuations in maternal energy balance, making the fetus in effect a “nearly perfect parasite.” This buffering would appear to be a reasonable adaptive response given the high risk of morbidity and mortality associated with low birth weight. However, a life-history approach leads to the prediction that maternal investment strategies in pregnancy should be geared toward maximizing lifetime reproductive success rather than simply the success of the current pregnancy, and by extension that maternal investment strategies should vary with reproductive value. The physiology of human pregnancy in fact appears to include a number of mechanisms that protect maternal energy resources from diversion to the fetus and preserve them for future reproductive events. These mechanisms include adjustment of blood flow to the uterus and perhaps minor adjustments in gestation length, although evidence for the latter is scant. Suggestions are made for ways of investigating these maternal options.     

Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells

The most commonly prescribed antidepressants, the serotonin (5-HT) selective reuptake inhibitors, increase 5-HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs-coupled 5-HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular-regulated kinase (ERK) and Akt pathways by Gs-coupled 5-HT7A receptors and Gq-coupled 5-HT2A receptors, which are co-expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5-HT2A receptor-mediated activation of the two pathways was found to be Ca2+-dependent. In contrast, 5-HT7A receptor-mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5-HT2A and 5-HT7A receptor agonists was found to be at least additive. Cell-permeable cAMP analogs mimicked 5-HT7A receptor agonists in enhancing 5-HT2A receptor-mediated activation of ERK and Akt. A role was identified for the cAMP-guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5-HT2A and 5-HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq- and Gs-coupled receptors.