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Martinez V.,Institute for Cell Dynamics and Biotechnology ICDB | Gerdtzen Z.P.,Institute for Cell Dynamics and Biotechnology ICDB | Andrews B.A.,Institute for Cell Dynamics and Biotechnology ICDB | Asenjo J.A.,Institute for Cell Dynamics and Biotechnology ICDB
Metabolic Engineering | Year: 2010

The HEK293 cell line has been used for the production of adenovirus vectors to be used in the potential treatment of alcoholism using a gene therapy strategy. Culture optimization and scale-up has been achieved by first adapting the cells to serum-free media and secondly by growing them in suspension. Adenovirus production after infection was increased, resulting in higher specific glucose consumption and lactate accumulation rates compared to the growth phase. We applied media design tools and Metabolic Flux Analysis (MFA) to compare the metabolic states of cells during growth and adenovirus production and to optimize culture media according to the metabolic demand of the cells in terms of glucose and glutamine concentrations. This allowed obtaining a higher maximum cell concentration and increased adenovirus production by minimizing the production of metabolites that can have an inhibitory effect on cell growth. We have proposed a stoichiometric equation for adenovirus synthesis. MFA results allowed determination of how these changes in composition affected the way cells distribute their nutrient resources during cell growth and virus production. Virus purification was successfully achieved using chromatography and Aqueous Two-Phase Systems (ATPS). © 2009 Elsevier Inc. All rights reserved.

Kahn O.I.,Drexel University | Sharma V.,Drexel University | Gonzalez-Billault C.,Institute for Cell Dynamics and Biotechnology ICDB | Baas P.W.,Drexel University
Molecular Biology of the Cell | Year: 2015

Kinesin-5 is a slow homotetrameric motor protein best known for its essential role in the mitotic spindle, where it limits the rate at which faster motors can move microtu-bules. In neurons, experimental suppression of kinesin-5 causes the axon to grow faster by increasing the mobility of microtubules in the axonal shaft and the invasion of microtubules into the growth cone. Does kinesin-5 act differently in dendrites, given that they have a population of minus end-distal microtubules not present in axons? Using rodent primary neurons in culture, we found that inhibition of kinesin-5 during various windows of time produces changes in dendritic morphology and microtubule organization. Specifically, dendrites became shorter and thinner and contained a greater proportion of minus end-distal microtu-bules, suggesting that kinesin-5 acting normally restrains the number of minus end-distal microtubules that are transported into dendrites. Additional data indicate that, in neurons, CDK5 is the kinase responsible for phosphorylating kinesin-5 at Thr-926, which is important for kinesin-5 to associate with microtubules. We also found that kinesin-5 associates preferentially with microtubules rich in tyrosinated tubulin. This is consistent with an observed accumulation of kinesin-5 on dendritic microtubules, as they are known to be less detyrosinated than axonal microtubules. © 2015 Kahn et al.

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