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1.
Lipopolysaccharide (LPS), the active component of bacterial endotoxin, caused no significant increase in ornithine decarboxylase (ODC) activity in serum-starved, Chinese hamster ovary fibroblasts. However, concurrent addition of LPS with 10% fetal bovine serum caused a synergistic 30 to 40-fold increase in enzyme activity as compared to the 10 to 20-fold increase seen after addition of serum alone. This synergism was not due to an alteration in the time course of enzyme induction after serum addition. The LPS-induced synergy of ODC induction by serum was inhibited by the concurrent addition of the specific LPS-antagonist, Polymyxin B. 相似文献
2.
Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca++ and Mg++ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity. 相似文献
3.
Fujiwara M Tsukada R Tsujinaga Y Takagi M 《Applied microbiology and biotechnology》2007,75(5):983-987
The effects of fish serum on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in
an adhesion culture of Chinese hamster ovary (CHO) cells DR1000L4N were investigated and compared with those of fetal calf
serum (FCS). Although fish serum did not stimulate the initial adhesion of CHO cells to culture dishes, it prompted cell growth
after cell adhesion with FCS for 24 h. The cell density in the fish serum medium reached 75% that in the FCS medium. Fish
serum promoted cell adhesion to and cell growth on collagen-coated dishes. The cell-specific production rate of hGM-CSF in
the fish serum medium on collagen-coated dishes was almost the same as that in the FCS medium. 相似文献
4.
Sodium butyrate (NaBu), which is widely used in recombinant Chinese hamster ovary cell (rCHO) cultures for high-level expression of therapeutic proteins, is known to induce apoptosis in a dose-dependent manner. Lately, the significance of autophagy has increased in the field of CHO cell culture due to the fact that autophagy is related to the programmed cell death mechanism. To determine the effect of NaBu on autophagy as well as apoptosis of rCHO cells, rCHO cells producing erythropoietin were subjected to NaBu treatment. NaBu treatment up to 5 mM increased cleaved forms of PARP, caspase-3, and Annexin V positive population, confirming the previous results that NaBu induces apoptosis. Concurrently, NaBu treatment increased the level of accumulation of the autophagic marker, LC3-II, independently of nutrient depletion, suggesting that NaBu induces autophagy. To elucidate the potential role of autophagy induced by NaBu, a representative autophagy inducer (rapamycin) or an inhibitor (bafilomycin A1) was added to cultures together with NaBu. It was found that autophagy had the potential role of a positive cell survival mechanism under NaBu treatment. Furthermore, gradual reduction in mitochondrial membrane potential/mass and recruitment of a mitophagy protein, Parkin, to the mitochondria were observed under NaBu treatment, suggesting that this positive function of autophagy might be mediated by the autophagic removal of damaged mitochondria. Taken together, autophagy was observed in rCHO cell culture under NaBu treatments and the results obtained here support the positive effects of autophagy induced by NaBu treatments. 相似文献
5.
Mitsuo Satoh Shinji Hosoi Seiji Sato 《In vitro cellular & developmental biology. Plant》1990,26(11):1101-1104
Summary The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or
aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells
increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular
proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide,
was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium.
In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although
amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases.
This work was supported by the management of the Research Association for Biotechnology as a part of the R&D of Basic Technology
for Future Industries sponsored by NEDO (New Energy and Industrial Technology Development Organization). 相似文献
6.
We determined the kinetics of the induction of chromosomal aberrations and micronuclei (MN) by mitomycin C (MMC, 0.1 µg/ml) in Chinese hamster ovary (CHO) cells treated with cytochalasin B (Cyt-B, 3 µg/ml). In cells treated with Cyt-B as well as with Cyt-B plus MMC the highest yield of binucleated cells was obtained 24 h after treatment. After 40 h of treatment with Cyt-B the frequency of MN in binucleated cells was significantly higher than that observed at previous times in the same cultures as well as in controls. In cultures treated with MMC the frequency of MN increased with time, reaching the highest value at 24 h. The frequency of chromosomal aberrations was also significantly higher in cells treated both with Cyt-B and Cyt-B plus MMC than in controls and exceeded that of MN in parallel cultures. These data confirm the capacity of MMC to induce chromosomal alterations in mammalian cells; in particular they indicate that Cyt-B is able to induce cytogenetic effects in CHO cells. Using immunofluorescence microscopy, after reaction with CREST antikinetochore antibodies, we found that in cells treated with Cyt-B or Cyt-B plus MMC the frequency of MN without kinetochore was, respectively, about 70 and 85%, indicating that under our experimental conditions MN originate mainly from acentric chromatid fragments. Present data suggest that the method based on the blockage of cytokinesis by Cyt-B normally used in the MN assay should be reconsidered. 相似文献
7.
Indomethacin inhibits cholera toxin-induced cyclic AMP accumulation in Chinese hamster ovary cells 总被引:1,自引:0,他引:1
Abstract Indomethacin was examined for its capacity to inhibit increases in adenosine-3',5'-monophosphate (cAMP) concentrations in Chinese hamster ovary (CHO) cells treated with cholera toxin. When added to the culture medium 1 h prior to cholera toxin (100 ng/ml), indomethacin (500 μg/ml) exhibited maximum protection against the typical increase in cAMP. Application of indomethacin at the same time as cholera toxin or up to 3 h after the toxin progressively decreased the drug's capacity to block further increases in cAMP. The drug appeared to block adenylate cyclase activity because addition of forskolin to drug-treated cells did not elicit a cAMP response. Binding of 125 I-labeled cholera toxin to indomethacin-treated cells was also reduced by at least 50%. These data indicate that indomethacin's inhibitory effect on cAMP formation in cholera toxin-treated cells could be explained by its capacity to alter adenylate cyclase activity and cholera toxin binding. 相似文献
8.
9.
Putrescine activates oxidative stress dependent apoptotic death in ornithine decarboxylase overproducing mouse myeloma cells 总被引:3,自引:0,他引:3
Accumulation of putrescine in ornithine decarboxylase overproducing cells provokes apoptotic death that is inhibited by 2-difluoromethylornithine, a specific inhibitor of ODC. The apoptotic process provoked by putrescine involves the release of cytochrome c from the mitochondria and activation of caspases cascades demonstrated by the cleavage of caspase-2, polyA-ribose polymerase (PARP), and proteolytic cleavage of the translation initiation factor 4G (eIF4G). The general caspases inhibitor BD-fmk inhibits PARP cleavage but not cell death. Aminoguanidine, an inhibitor of amine oxidases, inhibits the cleavage of PARP and cell death, whereas the antioxidant BHA inhibits PARP cleavage but not cell death. The intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) inhibits both PARP cleavage and cell death. Although the ability of BAPTA/AM to inhibit the induction of apoptosis may suggest that the accumulating putrescine stimulates the release of Ca2+, such a Ca2+ elevation was not observed. We suggest that the accumulation of putrescine leads to oxidative stress that causes cell death. 相似文献
10.
11.
12.
Hwang SJ Choi HH Kim KT Hong HJ Koh GY Lee GM 《Protein expression and purification》2005,39(2):14410-183
Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future. 相似文献
13.
David J. Robins 《Phytochemistry》1983,22(5):1133-1135
The decarboxylations of l-arginine, catalysed by arginine decarboxylase (EC 4.1.1.19) and of l-ornithine, catalysed by ornithine decarboxylase 相似文献
14.
Dongsheng Xu Aini Wan Lin Peng Yun Chen Yang He Jianfeng Yang 《Preparative biochemistry & biotechnology》2017,47(5):489-495
Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12?mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins. 相似文献
15.
Expression and purification of recombinant human Angiopoietin-1 produced in Chinese hamster ovary cells 总被引:1,自引:0,他引:1
Hwang SJ Kim HZ Koh GY Lee GM 《In vitro cellular & developmental biology. Animal》2007,43(5-6):162-167
Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1,
recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an
amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO
cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and
0.32 μM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using
an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous
multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind
to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying
the biological effects of exogenous rhAng1 in the future. 相似文献
16.
Diana Széliová Dmytro Iurashev David E Ruckerbauer Gunda Koellensperger Nicole Borth Michael Melcher Jürgen Zanghellini 《Biotechnology journal》2021,16(4):2000320
Chinese hamster ovary (CHO) cells are the most popular mammalian cell factories for the production of glycosylated biopharmaceuticals. To further increase titer and productivity and ensure product quality, rational system-level engineering strategies based on constraint-based metabolic modeling, such as flux balance analysis (FBA), have gained strong interest. However, the quality of FBA predictions depends on the accuracy of the experimental input data, especially on the exchange rates of extracellular metabolites. Yet, it is not standard practice to devote sufficient attention to the accurate determination of these rates. In this work, we investigated to what degree the sampling frequency during a batch culture and the measurement errors of metabolite concentrations influence the accuracy of the calculated exchange rates and further, how this error then propagates into FBA predictions of growth rates. We determined that accurate measurements of essential amino acids with low uptake rates are crucial for the accuracy of FBA predictions, followed by a sufficient number of analyzed time points. We observed that the measured difference in growth rates of two cell lines can only be reliably predicted when both high measurement accuracy and sampling frequency are ensured. 相似文献
17.
Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC
ornithine decarboxylase
- AdoMetDC
S-adenosylmethionine decarboxylase
- DFMO
difluoromethylornithine 相似文献
18.
19.
The effects of heat treatment and concentration of fish serum (FS) on cell growth and human granulocyte-macrophage colony-stimulating
factor (hGM-CSF) production in an adhesion culture of recombinant Chinese hamster ovary (CHO) cells, DR1000L4N, were investigated.
The addition of heat treated FS instead of non-heat-treated FS improved cell growth in terms of cell density, which reached
60% that in 10% fetal calf serum (FCS)-containing medium (FCS medium). A decrease in FS concentration from 10 to 1.25% markedly
increased cell density, which was 79% that in 10% FCS medium. The combination of heat treatment at 56 °C and the addition
of FS at a low concentration (1.25%) showed an additive effect on cell growth and resulted in the same cell density as that
in 10% FCS medium, whereas the hGM-CSF concentration in the culture using FS-containing medium (FS medium) was approximately
50% that in 10% FCS medium. The total lipid concentration in FS was more than three fold that in FCS. The effect of decreasing
FS concentration on cell growth may be due to the low lipid concentration in FS medium, because addition of the lipids extracted
from FS to 10% FCS and 1.25% FS media markedly decreased cell density. Consequently, the addition of heat-treated FS at low
concentrations to medium may be useful for the growth of CHO cells without FCS. 相似文献
20.
Hayter PM Curling EM Baines AJ Jenkins N Salmon I Strange PG Tong JM Bull AT 《Biotechnology and bioengineering》1992,39(3):327-335
A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma. 相似文献