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141.
Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-13C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and 13C-labeling dynamics of intracellular metabolites using non-stationary 13C-metabolic flux analysis (13C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins. 相似文献
142.
Kinases, representing almost 500 proteins in the human genome, are responsible for catalyzing the phosphorylation reaction
of amino acid residues at their targets. As the largest family of kinases, the protein tyrosine kinases (PTKs) have roles
in controlling the essential cellular activities, and their deregulation is generally related to pathologic conditions. The
recent efforts on identifying their signal transducer or mediator role in cellular signaling revealed the interaction of PTKs
with numerous enzymes of different classes, such as Ser/Thr kinases (STKs), glutathione transferases (GSTs), and receptor
tyrosine kinases (RTKs). In either regulation or enhancing the signaling, PTKs are determined in close interaction with these
enzymes, under specific cellular conditions, such as oxidative stress and inflammation. In this concept, intensive research
on thiol metabolizing enzymes recently showed their involvement in the physiologic functions in cellular signaling besides
their well known traditional role in antioxidant defense. The shared signaling components between PTK and GST family enzymes
will be discussed in depth in this research review to evaluate the results of recent studies important in drug targeting for
therapeutic intervention, such as cell viability, migration, differentiation and proliferation. 相似文献
143.
Ria Heinsbroek Jan van Brederode Gerrit van Nigtevecht John Kamsteeg 《Phytochemistry》1979,18(6):935-937
In petals of Silene dioica plants, an enzyme has been demonstrated which catalyses the transfer of the arabinose moiety of UDP-arabinose to the hydroxyl group on the 2″-position of the carbon-carbon bound glucose of isovitexin. The presence of this arabinosyltransferase activity is controlled by the dominant allele glA. Maximal activity takes place at pH 7.2–7.5; the reaction is stimulated by the divalent metal ions Mg and Mn. For optimal solubilization of the enzyme, Triton X-100 is necessary. Substrate specificity and kinetic behaviour have been investigated. 相似文献
144.
Jacques A Durr Mary Blankenship Satendra S Chauhan Michael W Pennington 《Journal of peptide science》2007,13(11):756-761
Iodination of the conserved 2-tyrosine (Tyr(2)) residue in the pressin and tocin rings of arginine- or lysine-vasopressin (AVP or LVP), and oxytocin, respectively, impairs binding to their respective receptors. Synthetic antagonists that have their Tyr(2) either replaced by another amino acid or irreversibly blocked by an O-methyl or O-ethyl ether, but have, instead, an iodinatable phenol moiety outside the pressin/tocin ring, are used for radiolabeling. We explored another approach to avoid iodinating Tyr(2) by capping this residue with a reversible O-acetyl group, incorporated during peptide synthesis. The O-acetyl-Tyr(2) LVP peptide, with a free iodinatable tyrosine attached to the epsilon-amine of 8-lysine, is iodinated at a neutral pH and purified by reverse-phase high-pressure liquid chromatography (HPLC) at an acidic pH, conditions under which the O-acetyl groups are stable. Deacetylation with hydroxylamine is selective, and leaves intact the disulfide bridge. The marked shortening of the HPLC retention time after deblocking produces a chemically homogeneous label, iodinated exclusively on the free tyrosine residue attached to the epsilon-amine of LVP. Hitherto, this (125)I labeled vasopressin agonist could be obtained only in low yield, via conjugation labeling with iodinated N-t-Boc-tyrosine succinimidyl ester. This fully reversible tyrosine protection strategy does not require special equipment, and retains the conserved Tyr(2), typical of vasopressin and oxytocin agonists. 相似文献
145.
146.
Tae-Jin?Kang So-Chon?Han Moon-Sik?YangEmail author 《Plant Cell, Tissue and Organ Culture》2005,81(2):165-174
The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants. 相似文献
147.
Saidi Y Finka A Chakhporanian M Zrÿd JP Schaefer DG Goloubinoff P 《Plant molecular biology》2005,59(5):697-711
The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool
for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using β-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 °C. In contrast, a short non-damaging heat-treatment at 38 °C rapidly induced
reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in
all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment,
allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3%
of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol
(BA) also induced GUS expression at 25 °C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in
the moss P. patens. 相似文献
148.
A.?L.?KhandazhinskayaEmail author M.?K.?Kukhanova M.?V.?Jasko 《Russian Journal of Bioorganic Chemistry》2005,31(4):352-356
N-(9-Fluorenylmethoxycarbonyl)-ω-aminoalkyl-, N-(9-fluorenylmethoxycarbonyl)-8-amino-3,6-dioxaoctyl, and N-[(9-fluorenylmethoxycarbonyl)-6-aminohexanoyl]-2-aminoethyl triphosphates were synthesized. All of them were shown to be the substrates of the calf thymus terminal deoxynucleotidyl transferase. Their substrate properties depend on the length and structure of the linker between the 9-fluorenylmethoxycarbonyl and triphosphate moieties.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 394–398.Original Russian Text Copyright © 2005 by Khandazhinskaya, Kukhanova, Jasko. 相似文献
149.
Novel triazole based inhibitors of Ras farnesyl transferase 总被引:1,自引:0,他引:1
Saha AK Liu L Simoneaux R DeCorte B Meyer C Skrzat S Breslin HJ Kukla MJ End DW 《Bioorganic & medicinal chemistry letters》2005,15(24):5407-5411
A novel series of potent inhibitors of Ras farnesyl transferase possessing a 1,2,4-triazole pharmacophore is described. These inhibitors were discovered from a parallel synthesis effort and were subsequently optimized to in vitro IC50 value of less than 1 nM. 相似文献
150.
Reduction of apoptosis through the mitochondrial pathway by the administration of acetyl-L-carnitine to mouse fibroblasts in culture 总被引:1,自引:0,他引:1
It is shown in literature that stress, such as deprivation of trophic factors and hypoxia, induces apoptosis in cultured cells and in tissues. In light of these results, we explored the possibility of protecting cells from programmed death by improving the metabolism of the mitochondrion. To this end, acetyl-L-carnitine was administered at various concentrations under conditions of serum deprivation. The choice of this drug was based on the accepted notion that acetyl-L-carnitine is able to stabilize mitochondrial membranes and to increase the supply of energy to the organelle. The results presented here indicate that the drug protects cells from apoptotic death: this is demonstrated by a lower positivity to the TUNEL reaction and by a strong reduction of the apoptotic DNA ladder in serum-deprived cells. The involvement of the mitochondrial apoptotic pathway was assessed by cytochrome C release and immunoreactivity to caspase 3. Moreover, acetyl-L-carnitine stimulates cell proliferation. 相似文献