Control of amino acid transport into Chinese hamster ovary cells.

Amino acid transporters (AATs) represent a key interface between the cell and its environment, critical for all cellular processes: Energy generation, redox control, and synthesis of cell and product biomass. However, very little is known about the activity of different functional classes of AATs in Chinese hamster ovary (CHO) cells, how they support cell growth and productivity, and the potential for engineering their activity and/or the composition of amino acids in growth media to improve CHO cell performance in vitro. In this study, we have comparatively characterized AAT expression in untransfected and monoclonal antibody (MAb)-producing CHO cells using transcriptome analysis by RNA-seq, and mechanistically dissected AAT function using a variety of transporter-specific chemical inhibitors, comparing their effect on cell proliferation, recombinant protein production, and amino acid transport. Of a possible 56 mammalian plasma membrane AATs, 16 AAT messenger RNAs (mRNAs) were relatively abundant across all CHO cell populations. Of these, a subset of nine AAT mRNAs were more abundant in CHO cells engineered to produce a recombinant MAb. Together, upregulated AATs provide additional supply of specific amino acids overrepresented in MAb biomass compared to CHO host cell biomass, enable transport of synthetic substrates for glutathione synthesis, facilitate transport of essential amino acids to maintain active protein synthesis, and provide amino acid substrates for coordinated antiport systems to maintain supplies of proteinogenic and essential amino acids.

A novel site of adult doublecortin expression: neuropeptide neurons within the suprachiasmatic nucleus circadian clock.

BACKGROUND: The mammalian suprachiasmatic nucleus (SCN) is composed of heterogeneous sub-groups of neurons that are organized into a neural system for the control of circadian physiology and behaviour. Molecular circadian 'clocks' are not an exclusive property of SCN neurons but the unique role of the SCN as a central integrative pacemaker is associated with specialized aspects of neuronal organization. Current studies are aimed at identifying the functional components of this hypothalamic integrative centre. RESULTS: In the present study we have identified and characterized a quite novel aspect of SCN neurobiology, doublecortin (DCX) protein expression within a defined group of adult rat SCN neurons. Adult neuronal DCX expression is surprising because this microtubule-associated protein (MAP) is generally a developmentally restricted component of immature, migrating neurons. We have also demonstrated for the first time that the SCN as a whole exhibits low expression of the neuronal differentiation marker NeuN. However, DCX is co-localized with NeuN in the ventral SCN, and also with neuropeptides; DCX is extensively co-localized with GRP and partially co-localized with VIP. CONCLUSION: The highly selective expression of DCX in the adult SCN compared with other hypothalamic and thalamic nuclei shows that this MAP is somewhat uniquely required in certain SCN neurons, perhaps contributing to a specific functional property of the brain's circadian clock nucleus. DCX may maintain a capacity for dynamic cellular plasticity that subserves daily alterations in SCN neuronal signalling.