Diversity in host clone performance within a Chinese hamster ovary cell line

Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc‐fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool‐produced Mab and etanercept (by N‐glycan ultra performance liquid chromatography (UPLC) and liquid chromatography ‐ tandem mass spectrometry (LC‐MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N‐glycan micro‐heterogeneity and etanercept N and O‐linked macro‐heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1187–1200, 2015     

Both the stimulation and inhibition of root hair growth induced by extracellular nucleotides in Arabidopsis are mediated by nitric oxide and reactive oxygen species

Root hairs secrete ATP as they grow, and extracellular ATP and ADP can trigger signaling pathways that regulate plant cell growth. In several plant tissues the level of extracellular nucleotides is limited in part by ectoapyrases (ecto-NTPDases), and the growth of these tissues is strongly influenced by their level of ectoapyrase expression. Both chemical inhibition of ectoapyrase activity and suppression of the expression of two ectoapyrase enzymes by RNAi in Arabidopsis resulted in inhibition of root hair growth. As assayed by a dose-response curve, different concentrations of the poorly hydrolysable nucleotides, ATPγS and ADPβS, could either stimulate (at 7.5–25 μM) or inhibit (at ≥ 150 μM) the growth rate of root hairs in less than an hour. Equal amounts of AMPS, used as a control, had no effect on root hair growth. Root hairs of nia1nia2 mutants, which are suppressed in nitric oxide (NO) production, and of atrbohD/F mutants, which are suppressed in the production of H₂O₂, did not show growth responses to applied nucleotides, indicating that the growth changes induced by these nucleotides in wild-type plants were likely transduced via NO and H₂O₂ signals. Consistent with this interpretation, treatment of root hairs with different concentrations of ATPγS induced different accumulations of NO and H₂O₂ in root hair tips. Two mammalian purinoceptor antagonists also blocked the growth responses induced by extracellular nucleotides, suggesting that they were initiated by a receptor-based mechanism.     

Domain formation by a Rhodococcus sp. biosurfactant trehalose lipid incorporated into phosphatidylcholine membranes

The study of the interaction of biosurfactants with biological membranes is of great interest in order to gain insight into the molecular mechanisms of their biological actions. In this work we report on the interaction of a bacterial trehalose lipid produced by Rhodococcus sp. with phosphatidylcholine membranes. Differential scanning calorimetry measurements show a good miscibility of the glycolipid in the gel state and immiscibility in the fluid state, suggesting domain formation. These domains have been visualized and characterized, for the first time, by scanning force microscopy. Incorporation of trehalose lipid into phosphatidylcholine membranes produces a small shift of the antisymmetric stretching band toward higher wavenumbers, as shown by FTIR, which indicates a weak increase in fluidity. The CO stretching band shows that incorporation of trehalose lipid increases the proportion of the dehydrated component in mixtures with the three phospholipids at temperatures below and above the gel to liquid-crystalline phase transition. This dehydration effect is also supported by data on the phospholipid PO stretching bands. Small-angle X-ray diffraction measurements show that in the samples containing trehalose lipid the interlamellar repeat distance is larger than in those of pure phospholipids. These results are discussed within the frame of trehalose lipid domain formation, trehalose lipid/phospholipid interactions and its relevance to membrane-related biological actions.     

Liver glyconeogenesis: a pathway to cope with postprandial amino acid excess in high-protein fed rats?

This paper provides molecular evidence for a liver glyconeogenic pathway, that is, a concomitant activation of hepatic gluconeogenesis and glycogenesis, which could participate in the mechanisms that cope with amino acid excess in high-protein (HP) fed rats. This evidence is based on the concomitant upregulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression, downregulation of glucose 6-phosphatase catalytic subunit (G6PC1) gene expression, an absence of glucose release from isolated hepatocytes and restored hepatic glycogen stores in the fed state in HP fed rats. These effects are mainly due to the ability of high physiological concentrations of portal blood amino acids to counteract glucagon-induced liver G6PC1 but not PEPCK gene expression. These results agree with the idea that the metabolic pathway involved in glycogen synthesis is dependent upon the pattern of nutrient availability. This nonoxidative glyconeogenic disposal pathway of gluconeogenic substrates copes with amino excess and participates in adjusting both amino acid and glucose homeostasis. In addition, the pattern of PEPCK and G6PC1 gene expression provides evidence that neither the kidney nor the small intestine participated in gluconeogenic glucose production under our experimental conditions. Moreover, the main glucose-6-phosphatase (G6Pase) isoform expressed in the small intestine is the ubiquitous isoform of G6Pase (G6PC3) rather than the G6PC1 isoform expressed in gluconeogenic organs.     

Synthesis and anti-inflammatory/antioxidant activities of some new ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-yl)-2-propen-1-one derivatives and of their 4,5-dihydro-(1H)-pyrazole analogues

We report the synthesis, anti-inflammatory and antioxidant activities of novel ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-yl)-2-propen-1-one derivatives and of their 4,5-dihydro-(1H)-pyrazole analogues. The tested compounds inhibit the carrageenin-induced rat paw edema (4.5-56.1%) and present important scavenging activities. Compound 2a is the most potent (56.1%) in the in vivo experiment and exhibits promising in vitro inhibition of soybean lipoxygenase (IC(50)<1microM).     

Novel series of substituted biphenylmethyl urea derivatives as MCH-R1 antagonists for the treatment of obesity

We have designed and synthesized two novel series of MCH-R1 antagonists based on a substituted biphenylmethyl urea core. SAR was explored, suggesting that optimal binding with the receptor was achieved when the biphenylmethyl group and the linker were substituted on the same nitrogen of the urea moiety. Compound 1-(3'-cyano-4-biphenylmethyl)-3-(2-hydroxy-1,1-dimethylethyl)-1-{2-[1-(4-methylbenzyl)-4-piperidinyl]ethyl}urea 2t showed the best antagonist binding activity to the MCH-R1 with a 43 nM K(i).     

Age-related increase in xanthine oxidase activity in human plasma and rat tissues

This study assessed the role of xanthine oxidase in vascular ageing. A positive correlation between xanthine oxidase activity and age was found in human plasma. Similar results were found in rat plasma. Xanthine oxidase expression and activity in homogenates from the aortic wall were significantly higher in samples from old rats than in their young counterparts (p < 0.01). In rat skeletal muscle homogenates both xanthine oxidase expression and activity showed a similar age-related profile. Superoxide production by xanthine oxidase in aortic rings was higher in aged rats. Uric acid, the final product of xanthine oxidase has been proposed as a risk factor for coronary heart disease and an independent marker of worse prognosis in patients with moderate-to-severe chronic heart failure. These results give a possible explanation for this correlation and underscore the role of xanthine oxidase in ageing.     

Acclimation to light in seedlings of <Emphasis Type="Italic">Quercus petraea</Emphasis> (Mattuschka) Liebl. and <Emphasis Type="Italic">Quercus pyrenaica</Emphasis> Willd. planted along a forest-edge gradient

Photosynthetic acclimation of two co-occurring deciduous oaks (Quercus petraea and Quercus pyrenaica) to a natural light gradient was studied during one growing season. In the spring of 2003, 90 seedlings per species were planted along a transect resulting from a dense Pinus sylvestris stand, an adjacent thinned area and a 10-m-wide firebreak (16.5–60.9% Global Site Factor (GSF)). In two dates of the following summer, we measured leaf gas exchange, carboxylation efficiency (CE), chlorophyll and nitrogen content, light–response curves of chlorophyll a fluorescence parameters, and leaf mass per area (LMA). Summer was mild, as evidenced by leaf predawn water potential (Ψ_pd), which reduced the interactive effect of water stress on the response of seedlings to light. Q. pyrenaica had higher LMA, CE, stomatal conductance (g _{s max}) and photosynthesis per unit area than Q. petraea at all growth irradiances. , LMA, g _{s max} and electron transport rate (ETR) all increased with light availability (GSF) in a similar fashion in both species. Light had also a clear effect on the organization of Photosystem II (PS II), as deduced by chlorophyll a fluorescence curves. Chlorophyll concentration (Chl_m) decreased with increasing light availability in Q. pyrenaica but it did not in Q. petraea. Seedlings of Q. petraea acclimated to higher irradiances showed a greater non-photochemical quenching (NPQ) than those of Q. pyrenaica. This suggests a higher susceptibility to high light in Q. petraea, which would be consistent with a better adaptation to shade, inferred from the lower LMA or the lower rate of photosynthesis.