Optimization of an acoustic cell filter with a novel air-backflush system

Increasing worldwide demand for mammalian cell production capacity will likely be partially satisfied by a greater use of higher volumetric productivity perfusion processes. An important additional component of any perfusion system is the cell retention device that can be based on filtration, sedimentation, and/or acoustic technologies. A common concern with these systems is that pumping and transient exposure to suboptimal medium conditions may damage the cells or influence the product quality. A novel air-backflush mode of operating an acoustic cell separator was developed in which an injection of bioreactor air downstream of the separator periodically returned the captured cells to the reactor, allowing separation to resume within 20 s. This mode of operation eliminated the need to pump the cells and allows the selection of a residence time in the separator depending on the sensitivity of the cell line. The air-backflush mode of operating a 10L acoustic separator was systematically tested at 10(7) cells/mL to define reliable ranges of operation. Consistent separation performance was obtained for wide ranges of cooling airflow rates from 0 to 15 L/min and for backflush frequencies between 10 and 40 h(-1). The separator performance was optimized at a perfusion rate of 10 L/day to obtain a maximum separation efficiency of 92 +/- 0.3%. This was achieved by increasing the power setting to 8 W and using duty cycle stop and run times of 4.5 and 45 s, respectively. Acoustic cell separation with air backflush was successfully applied over a 110 day CHO cell perfusion culture at 10(7) cells/mL and 95% viability.     

Genome of Azotobacter vinelandii: counting of chromosomes by utilizing copy number of a selectable genetic marker

Studies utilizing several physical, biochemical and spectroscopic methods have suggested that Azotobacter vinelandii contains multiple copies (40–80) of its chromosome per cell, whereas genetic analysis indicated that these cells function like haploid cells. To further verify if A. vinelandii indeed contains 40–80 copies of its chromosome per cell, we have developed an in vivo chromosome counting technique. The basic principle of this technique is to introduce the same genetic marker on the chromosome and on an extrachromosomal element of known copy number into the bacterium. The copy number of the chromosome can be determined by comparing the intensity of the hybridization signal generated by the DNA fragment carrying the chromosomal marker with that of the extrachromosomal marker when the total DNA isolated from this strain is hybridized with a probe made of the same genetic marker DNA. To do this we used an A. vinelandii BG102 strain which carries a kanamycin resistance marker gene integrated into the nifY locus on its chromosome(s). The plasmids pRK293 and pKT230, which can replicate in A. vinelandii and carry the kanamycin resistance gene (similar to the one present on the chromosome of A. vinelandii BG102), served as the extrachromosomal elements with known copy number. Southern blotting and hybridization analysis of the total DNA, isolated from A. vinelandii BG102 containing these plasmids, with a probe made of the kanamycin resistance gene clearly indicated that the copy number of A. vinelandii chromosome is slightly lower than the copy number of the low-copy plasmid pRK293 and about 21-fold lower than the copy number of the high copy plasmid pKT230. We believe that this In vivo chromosome counting technique can be used for determination of the copy number of the chromosome in other cells with appropriate modifications in the nature of the extrachromosomal element and the genetic marker.     

Oligomerization of opioid receptors

Opioid receptors belong to the family of G-protein-coupled receptors characterized by their seven transmembrane domains. The activation of these receptors by agonists such as morphine and endogenous opioid peptides leads to the activation of inhibitory G-proteins followed by a decrease in the levels of intracellular cAMP. Opioid receptor activation is also associated with the opening of K(+) channels and the inhibition of Ca(2+) channels. A number of investigations, prior to the development of opioid receptor cDNAs, suggested that opioid receptor types interacted with each other. Early pharmacological studies provided evidence for the probable interaction between opioid receptors. More recent studies using receptor selective antagonists, antisense oligonucleotides, or animals lacking opioid receptors further suggested that interactions between opioid receptor types could modulate their activity. We examined opioid receptor interactions using biochemical, biophysical, and pharmacological techniques. We used differential epitope tagging and selective immunoisolation of receptor complexes to demonstrate homotypic and heterotypic interactions between opioid receptor types. We also used the proximity-based bioluminescence resonance energy transfer assay to explore opioid receptor-receptor interactions in living cells. In this article we describe the biochemical and biophysical methods involved in the detection of receptor dimers. We also address some of the concerns and suggest precautions to be taken in studies examining receptor-receptor interactions.     

Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein

The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length lambdaCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal alpha-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the beta-beta interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the alpha-RNAP of the pTRG vector and lambdaCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the beta-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the beta-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the beta-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein-protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.     

Accelerating perfusion process optimization by scanning non-steady-state responses

Perfusion processes provide consistent culture conditions, high productivity and low product residence times. However, process development can be slow due to the 1 week or more required to reach each steady state. The objective of this work was to accelerate process development in perfusion cultures by scanning non-steady-state transient responses to qualitatively predict steady-state performance. The method was tested using a shift in temperature every 3 days, scanned down by steps of 2 degrees C from 37 degrees C to 31 degrees C, then scanned up to 37 degrees C. Higher t-PA concentrations were predicted at lower temperatures, confirmed by subsequent pseudo-steady-state results. In most cases, transient values on the 3rd day were in close concordance with pseudo-steady-state values. To further accelerate process development, transient scanning was applied to small-scale, non-instrumented cultures. Similar results were obtained, although quantitative t-PA values were 15-30 times lower than in high cell density perfusion cultures. The method was further explored by investigating 1 day transient shifts in temperature where more variability was observed, suggesting that the cells were still adapting to the new environment. Nonetheless, the overall response again qualitatively predicted the pseudo-steady-state temperature response. Use of transient scanning in conjunction with pseudo-steady-state verification and refinement of optimal results could reduce process development time to a third or less of comparable steady-state-based optimization.     

Regulation of the selenoprotein SelS by glucose deprivation and endoplasmic reticulum stress - SelS is a novel glucose-regulated protein

SelS is a newly identified selenoprotein and its gene expression is up-regulated in the liver of Psammomys obesus after fasting. We have examined whether SelS is regulated by glucose deprivation and endoplasmic reticulum (ER) stress in HepG2 cells. Glucose deprivation and the ER stress inducers tunicamycin and thapsigargin increased SelS gene expression and protein content several-fold in parallel with glucose-regulated protein 78. The overexpression of SelS increased Min6 cell resistance to oxidative stress-induced toxicity. These results indicate that SelS is a novel member of the glucose-regulated protein family and its function is related to the regulation of cellular redox balance.