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1.
P. C. Huang Betty Smith Patricia Bohdan Alice Corrigan 《Biological trace element research》1980,2(3):211-220
Addition of zinc lowers the toxicity level of cadmium in cultured CHO cells. Cell survival and protein synthesis were used
to measure the cellular toxicity of cadmium.109Cd was used to measure cadmium uptake by the cells. The results suggest that these class IIB transition metals, zinc and cadmium,
share a common transport mechanism. Thus, the antagonism appears to involve a reduction in the influx of cadmium due to the
presence of zinc. 相似文献
2.
Stable mutants resistant to pactamycin (PacR), a polypeptide chain initiation inhibitor, have been selected in a single step in Chinese hamster ovary (CHO) cells. The sensitivity of protein synthesis in mutant cell extracts to pactamycin indicates that resistance involves an alteration in the permeability of this drug. The failure of PacR mutants to show cross-resistance to other compounds provides further indication that the lesion is presumably specific for pactamycin. Cell hybrids formed between PacR × PacS lines show intermediate sensitivity towards pactamycin, suggesting that the PacR lesion behaves codominantly under these conditions. In the presence of subinhibitory concentrations of pactamycin, CHO cells, which are normally short, polygonal and disoriented, became greatly elongated and aligned themselves in parallel fashion to produce highly oriented colony morphologies, reminiscent of normal diploid fibroblasts. This effect of pactamycin on cellular morphology was seen much more clearly with the PacR mutants, although somewhat higher concentrations of the drug were required to produce this change. 相似文献
3.
CHO cell aggregation induced by fibronectin-coated beads. Differences between wild-type and adhesion-variant cells 总被引:1,自引:0,他引:1
ADvF11 (F11), a Chinese Hamster Ovary (CHO) cell variant, is defective in its ability to adhere to fibronectin (Fn)-coated substrata but will adhere to substrata coated with poly-L-lysine, conA or extracellular matrix (ECM) [1]. We have observed that both F11 and CHO wild-type (WT) cells were able to bind 3H-Fn beads in a similar manner; however, only WT cells and not F11 cells aggregate in the presence of Fn beads. Both cell types aggregated similarly in the presence of lectins. Fn-bead-mediated aggregation was blocked by low temperature and aggregation did not occur when formaldehyde-fixed WT cells were used. Colchicine, tetracaine and cytochalasin B were not effective in blocking aggregation induced by Fn beads. These results suggest that: 1. Both WT and F11 cells have surface membrane-binding sites for Fn. 2. The aggregation defect in F11 cells is distal to the initial interaction between the cell surface and Fn, but proximal to the cytoskeletal rearrangements required for cell adhesion. 相似文献
4.
The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. It provides precursors for the biosynthesis of nucleotides and contributes to the production of reducing power in the form of NADPH. It has been hypothesized that mammalian cells may contain a hidden reaction in PPP catalyzed by transketolase-like protein 1 (TKTL1) that is closely related to the classical transketolase enzyme; however, until now there has been no direct experimental evidence for this reaction. In this work, we have applied state-of-the-art techniques in 13C metabolic flux analysis (13C-MFA) based on parallel labeling experiments and integrated flux fitting to estimate the TKTL1 flux in CHO cells. We identified a set of three parallel labeling experiments with [1-13C]glucose+[4,5,6-13C]glucose, [2-13C]glucose+[4,5,6-13C]glucose, and [3-13C]glucose+[4,5,6-13C]glucose and developed a new method to measure 13C-labeling of fructose 6-phosphate by GC-MS that allows intuitive interpretation of mass isotopomer distributions to determine key fluxes in the model, including glycolysis, oxidative PPP, non-oxidative PPP, and the TKTL1 flux. Using these tracers we detected a significant TKTL1 flux in CHO cells at the stationary phase. The flux results suggest that the main function of oxidative PPP in CHO cells at the stationary phase is to fuel the TKTL1 reaction. Overall, this study demonstrates for the first time that carbon atoms can be lost in the PPP, by means other than the oxidative PPP, and that this loss of carbon atoms is consistent with the hypothesized TKTL1 reaction in mammalian cells. 相似文献
5.
Transport and accumulation of 2-deoxy-D-glucose in wild-type and hexokinase-deficient cultured Chinese-hamster ovary (CHO) cells.
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Hexokinase-deficient mutants and wild-type Chinese-hamster ovary cells have been used to investigate the role of hexokinase in uptake and accumulation of 2-D-deoxyglucose (2-dGlc). The evidence for a specific sugar transport system in both types of cells is that there is similar saturable phloretin-sensitive uptake of 2-dGlc and 3-O-methyl-D-glucose (3-OMG) in both types of cell. In wild-type cells, 2-dGlc is accumulated to a tissue:medium ratio of 10- and in the mutant only 3-fold; 3-OMG is not accumulated by either mutant or wild-type cells. The evidence that hexokinase affects the membrane transport process is that the rate of exit of free 2-dGlc from wild-type cells is 5-fold less than from mutant cells, whereas there is no difference in the rate of loss of 3-OMG between mutant and wild-type cells. 相似文献
6.
W. E. C. Bradley 《Experimental cell research》1980,129(2)
The persistence of individual hypoxanthine phosphoribosyltransferase (HPRT)-deficient cells in small populations of mutagenized CHO was examined. Most of these variants were unstable with progressive cultivation in non-selective medium (α) before exposure to the selective agent, thioguanine (TG), but after selection virtually all resistant colonies were stable. The role of cell density in this effect was assessed by shifting sister cultures of low-density populations to high and then back to low density in α-medium and measuring cloning efficiency (CE) in TG after each shift. The high density cells almost always had a lower CE in TG than their low density siblings, indicating a relative loss of TG resistance. When they were passed again at low density, the higher CE of the sister culture was usually not reacquired. These variants therefore appeared to be sensitive to a density-dependent reversal of phenotype. This interpretation was verified by growing sister cultures of biochemically marked, mutagenized CHO cells in TG for 3 days. The resistant colonies were then grown in α-medium and challenged by co-incubating colonies of one dish with wild-type (WT) unmarked cells immediately and those of the sister dish with WT after various periods in α-medium. Most TG-resistant colonies underwent some degree of stable reversal to the HPRT+ phenotype when challenged immediately, but their sister colonies, tested at later times, became insensitive to this effect over periods ranging up to 30 days or more after mutagenesis. 相似文献
7.
Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance. 相似文献
8.
A cadmium-resistant variant of the Chinese hamster (CHO) cell with increased metallothionein induction capacity 总被引:12,自引:0,他引:12
C. E. Hildebrand R. A. Tobey E. W. Campbell M. D. Enger 《Experimental cell research》1979,124(2):237-246
The toxic trace metal Cd2+ has been used to select a variant (designated Cdr) of the Chinese hamster cell (line CHO) resistant to the growth-inhibitory and cytotoxic effects of Cd2+. Resistance of the Cdr cell to Cd2+-mediated cytotoxicity is not due to a decreased capability of the Cdr cell to accumulate Cd2+ since Cd2+ uptake in the Cdr cell is indistinguishable from that in the CHO cell at both toxic and subtoxic Cd2+ exposures. Comparison of the relative capacities of these two cell types to induce specific low molecular weight Cd2+-binding proteins (metallothioneins) reveals that the Cdr cell has an increased capacity to induce metallothionein and to sequester intracellular Cd2+ in metallothioneins. These results suggest that the greater competence of the Cdr cell to induce metallothionein is a major factor in the Cd2+-resistant phenotype of the variant. 相似文献
9.
Summary Cadmium and zinc biosorption byChlorella homosphaera cells were tested under laboratory conditions, in a range of concentrations from 0.5 to 14.0 mg/l. The results indicated two distinct phases for cadmium biosorption: a rapid phase probably associated with metal adsorption around the cell wall and a slower phase associated with the metal transport into the interior of the cells. For zinc biosorption these phases were not well defined probably due to the metabolic use of this metal by the cells. 相似文献
10.
Ultraviolet (UV) irradiation produces DNA photoproducts that are blocks to DNA replication by normal replicative polymerases. A specialized, damage-specific, distributive polymerase, Pol H or Pol h, that is the product of the hRad30A gene, is required for replication past these photoproducts. This polymerase is absent from XP variant (XP-V) cells that must employ other mechanisms to negotiate blocks to DNA replication. These mechanisms include the use of alternative polymerases or recombination between sister chromatids. Replication forks arrested by UV damage in virus transformed XP-V cells degrade into DNA double strand breaks that are sites for recombination, but in normal cells arrested forks may be protected from degradation by p53 protein. These breaks are sites for binding a protein complex, hMre11/hRad50/Nbs1, that colocalizes with H2AX and PCNA, and can be visualized as immunofluorescent foci. The protein complexes need phosphorylation to activate their DNA binding capacity. Incubation of UV irradiated XP-V cells with the irreversible kinase inhibitor wortmannin, however, increased the yield of Mre11 focus-positive cells. One interpretation of this observation is that two classes of kinases are involved after UV irradiation. One would be a wortmannin-resistant kinase that phosphorylates the Mre11 complex. The other would be a wortmannin-sensitive kinase that phosphorylates and activates the p53/large T in SV40 transformed XP-V cells. The sensitive class corresponds to the PI3-kinases of ATM, ATR, and DNA-PK, but the resistant class remains to be identified. Alternatively, the elevated yield of Mre11 foci positive cells following wortmannin treatment may reflect an overall perturbation to the signaling cascades regulated by wortmannin-sensitive PI3 related kinases. In this scenario, wortmannin could compromise damage inducible-signaling pathways that maintain the stability of stalled forks, resulting in a further destabilization of stalled forks that then degrade, with the formation of DNA double strand breaks. 相似文献
11.
E Carpenè G Crisetig P Cortesi G Serrazanetti 《Bollettino della Società italiana di biologia sperimentale》1979,55(12):1217-1223
The accumulation of Cadmium in Mytilus galloprovincialis during a 18 days exposure period was studied. Cadmium content of the mantle, viscera, gills increased throughout the time. The study of the distribution of Cd in homogenates of mussels chronically intoxicated has shown that the metal is principally bound to low mol. wt. proteins similar to metallothioneins. Mytilus galloprovincialis metallothioneins are also comparable to the vertebrate proteins, suggesting a biological function ubiquitous in the living world. 相似文献
12.
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
13.
Banfalvi G Gacsi M Nagy G Kiss ZB Basnakian AG 《Apoptosis : an international journal on programmed cell death》2005,10(3):631-642
CHO cells were grown in the presence of 1 M CdCl2 and subjected to ATP-dependent replicative DNA synthesis after permeabilization. By decreasing the density of the cell culture replicative DNA synthesis was diminishing. At higher than 2 × 106 cell/ml concentration Cd had virtually no effect on the rate of DNA replication. Growth at higher cell concentrations could be supressed by increasing Cd concentration. After Cd treatment cells were synchronized by counterflow centrifugal elutriation. Cadmium toxicity on cell growth in early and mid S phase led to the accumulation of enlarged cells in late S phase. Flow cytometry showed increased cellular and nuclear sizes after Cd treatment. As the cells progressed through the S phase, 11 subpopulations of nuclear sizes were distinguished. Apoptotic chromatin changes were visualized by fluorescent microscopy in a cell cycle dependent manner. In the control untreated cells the main transitory forms of chromatin corresponded to those we have published earlier (veil-like, supercoiled chromatin, fibrous, ribboned structures, chromatin strings, elongated prechromosomes, precondensed chromosomes). Cadmium treatment caused: (a) the absence of decondensed veil-like structures and premature chromatin condensation in the form of apoptotic bodies in early S phase (2.2–2.4 average C-value), (b) the absence of fibrous structures, the lack of supercoiled chromatin, the appearance of uncoiled ribboned chromatin and perichromatin semicircles, in early mid S phase (2.5–2.9 C), (c) the presence of perichromatin fibrils and chromatin bodies in mid S phase (2.9–3.2 C), (d) early intra-nuclear inclusions, elongated forms of premature chromosomes, the extrusion and rupture of nuclear membrane later in mid S phase (3.3–3.4 C), (e) the exclusion of chromatin bodies and the formation of clusters of large-sized perichromatin granules in late S phase (3.5–3.8 C) and (f) large extensive disruptions and holes in the nuclear membrane and the clumping of incompletely folded chromosomes (3.8–4. C). 相似文献
14.
Environmental exposure to cadmium (Cd) links to neurodegenerative disorders. Autophagy plays an important role in controlling cell survival/death. However, how autophagy contributes to Cd's neurotoxicity remains enigmatic. Here, we show that Cd induced significant increases in autophagosomes with a concomitant elevation of LC3-II and p62 in PC12 cells and primary neurons. Using autophagy inhibitor 3-MA, we demonstrated that Cd-increased autophagosomes contributed to neuronal apoptosis. Impairment of Cd on autophagic flux was evidenced by co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells. This is further supported by the findings that administration of chloroquine (CQ) potentiated the basic and Cd-elevated LC3-II and p62 levels, autophagosome accumulation and cell apoptosis, whereas rapamycin relieved the effects in the cells in response to Cd. Subsequently, we noticed that Cd evoked the phosphorylation of Akt and BECN1. Silencing BECN1 and especially expression of mutant BECN1 (Ser295A) attenuated Cd-increased autophagosomes and cell death. Of note, inhibition of Akt with Akt inhibitor X, or ectopic expression of dominant negative Akt (dn-Akt), in the presence or absence of 3-MA, significantly alleviated Cd-triggered phosphorylation of Akt and BECN1, autophagosomes, and apoptosis. Importantly, we found that Cd activation of Akt functioned in impairing autophagic flux. Collectively, these results indicate that Cd results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells. Our findings underscore that inhibition of Akt to improve autophagic flux is a promising strategy against Cd-induced neurotoxicity and neurodegeneration. 相似文献
15.
M A Nedelman R C Elder K Tepperman 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1989,191(2):179-186
Gold sodium thiomalate was incubated with one cadmium-sensitive cell line and two cadmium-resistant variants. The resistant lines have been reported to synthesize metallothionein (MT) in response to both cadmium and zinc, whereas the sensitive line does not. All cell lines showed a dose-dependent inhibition of growth as a result of gold sodium thiomalate treatment. However, daily comparisons of cell numbers indicate that the cadmium-resistant lines actually increase in number at the highest gold concentrations, whereas numbers of cells in the nonresistant line decrease. MT biosynthesis was measured by monitoring the incorporation of [35S )cysteine into low molecular weight protein. None of the cells synthesized MT in response to gold. When incubated with both zinc and gold, MT was synthesized by both of the cadmium resistant lines; however, the amount of MT synthesized was reduced in the presence of gold which appears to inhibit the uptake of [35S]cysteine by all the cell lines. Although MT is synthesized in the presence of zinc and gold sodium thiomalate, the MT does not have a significant effect on the ability of these cells to withstand high concentrations of gold. 相似文献
16.
17.
With the increasingly competitive commercial production of target proteins by hybridoma and genetically engineered cells,
there is an urgent requirement for biosensors to monitor and control on-line and in real time the growth of cultured cells.
Since growth is accompanied by an enthalpy change, heat dissipation measured by calorimetry could act as an index for metabolic
flow rate. Recombinant CHO cell suspensions producing interferon-γ were pumped to an on-line flow calorimeter. The results
showed that an early reflection of metabolic change is size-specific heat flux obtained from dividing heat flow rate by the
capacitance change of the cell suspension, using the on-line probe of a dielectric spectroscope. Comparison of heat flux with
glucose and glutamine fluxes indicated that the former most accurately reflected decreased metabolic activity. Possibly this
was due to accumulation of lactate and ammonia resulting from catabolic substrates being used as biosynthetic precursors.
Thus, the heat flux probe is an ideal on-line biosensor for fed-batch culture. A stoichiometric growth reaction was formulated
and data for material and heat fluxes incorporated into it. This showed that cell demand for glucose and glutamine was in
the stoichiometric ratio of ∼3:1 rather than the ∼5:1 in the medium. It was demonstrated that the set of stoichiometric coefficients
in the reaction were related through the extent of reaction (advancement) to overall metabolic activity (flux). The fact that
this approach can be used for medium optimisation is the basis for an amino-acid-enriched medium which improved cell growth
while decreasing catabolic fluxes.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
18.
The plant cytoskeleton orchestrates such fundamental processes in cells as division, growth and development, polymer cross-linking, membrane anchorage, etc. Here, we describe the influence of Cd2+, Ni2+, Zn2+, and Cu2+ on root development and vital organization of actin filaments into different cells of Arabidopsis thaliana line expressing GFP-FABD2. CdSO4, NiSO4, CuSO4, and ZnSO4 were used in concentrations of 5–20 µM in this study. It was found that Cd, Ni, and Cu cause dose-dependent primary root growth inhibition and alteration of the root morphology, whereas Zn slightly stimulates root growth and does not affect the morphology of Arabidopsis roots. This growth inhibition/stimulation correlated with the various sensitivities of microfilaments to Cd, Ni, Cu, and Zn action. It was established that Cd, Ni, and Cu affected predominantly the actin filaments of meristematic cells. Cells of transition and elongation zones demonstrated strong actin filament sensitivity to Cd and Cu. Microfilaments of elongating root cells were more sensitive to Ni and Cu. Although Cd, Ni, and Cu stimulated root hair growth after long-term treatment, actin filaments were destroyed after 1 h exposure with these metals. Zn did not disrupt native actin filament organization in root cells. Thus, our investigation shows that microfilaments act as sensitive cellular targets for Cd, Ni, and Cu. More data on effects on native actin filaments organization would contribute to a better understanding of plant tolerance mechanisms to the action of these metals. 相似文献
19.
Susan C Burleigh Teun van de Laar Corné JM Stroop Wout MJ van Grunsven Niaobh O’Donoghue Pauline M Rudd Gavin P Davey 《BMC biotechnology》2011,11(1):1-17
Background
The photorespiratory nitrogen cycle in C3 plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO2. Because of the loss of CO2 and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C3 plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.Results
When grown in ambient air, but not in elevated CO2, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.Conclusions
Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars. 相似文献20.