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A Chinese hamster ovary (CHO) cell line that expresses human erythropoietin (huEPO) was in a 2-L Cytopilot fluidized-bed bioreactor with 400 mL macroporous Cytoline-1 microcarriers and a variable perfusion rate of serum-free and protein-free medium for 48 days. The cell density increased to a maximum of 23 x 10(6) cells/mL, beads on day 27. The EPO concentration increased to 600 U/mL during the early part of the culture period (on day 24) and increased further to 980 U/mL following the addition of a higher concentration of glucose and the addition of sodium butyrate. The EPO concentration was significantly higher (at least 2x than that in a controlled stirred-tank bioreactor, in a spinner flask, or in a stationary T-flask culture. The EPO accumulated to a total production of 28,000 kUnits over the whole culture period. The molecular characteristics of EPO with respect to size and pattern of glycosylation did not change with scale up. The pattern of utilization and production of 18 amino acids was similar in the Cytopilot culture to that in a stationary batch culture in a T-flask. The concentration of ammonia was maintained at a low level (< 2 mM) over the entire culture period. The specific rate of consumption of glucose, as well as the specific rates of production of lactate and ammonia, were constant throughout the culture period indicating a consistent metabolic behavior of the cells in the bioreactor. These results indicate the potential of the Cytopilot bioreactor culture system for the continuous production of a recombinant protein over several weeks. 相似文献
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The action of adrenal glucocorticoids on the level of liver beta-lipoprotein (LP) production was studied. Their effect was verified by studying LP synthesis and release from liver after the administration of various doses of glucocorticoids, after the administration of ACTH and in cases in which the effect of glucocorticoids was precluded by adrenalectomy (in vivo and in vitro). Adrenal glucocorticoids were found to limit triglyceride release in the form of beta-LP from the liver into the blood stream during the first 6 hours after partial hepatectomy (PHE). A direct study of beta-LP synthesis in the liver tissue showed that glucocorticoids inhibit liver lipid secretion by interfering with the process of beta-LP release by the liver rather than by influencing actual beta-LP synthesis in the liver. 相似文献
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To examine the influence of maternal lymphocyte factors on the immune responses in offspring in early life, antibody production in neonates born to either normal or lymphocyte-deficient mothers was analyzed. Recombination activating gene (Rag)-2(+/-) mouse neonates born to Rag-2(+/+), Rag-2(+/-)or Rag-2(-/-)mothers were injected with goat anti-mouse IgD antiserum, and IgE and IgG(1) production was evaluated. The levels of IgE and IgG(1) were higher in the pups born to Rag-2(+/+)and Rag-2(+/-) dams than to lymphocyte-deficient Rag-2(-/-) dams. The enhanced antibody production in the former compared with the latter neonates was also found following immunization with ovalbumin or TNP-Ficoll. Thus, the presence of maternal lymphocyte factors was suggested in neonates that augmented antigen-specific antibody production in both T cell-dependent and -independent pathways. A reduction in antibody production was observed in normal neonates when they were foster-nursed by Rag-2(-/-) mothers. Thus, the maternal lymphocyte factors enhancing the immune responses in newborns were shown to be present in breast-milk. 相似文献
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C H Kennedy J M Dyer D F Church G W Winston W A Pryor 《Biochemical and biophysical research communications》1989,160(3):1067-1072
Eleven peroxides have been tested to determine if there is a correlation between tumor-promoting activity and the ability to stimulate radical production in mitochondria. When non-respiring rat liver mitochondria are treated with these peroxidic compounds in the presence of DMPO, ESR signals are observed from the spin trapping of carbon- and oxygen-centered radicals in the case of 4 of the 7 peroxides that are known to be tumor promoters. Enhancement of carbon-centered radical production is observed in the presence of respiratory substrate. Thus there does not appear to be a correlation between tumor-promoting activity of peroxidic compounds and radical production in mitochondria. Oxidants can act as promoters either by 1- or 2-electron oxidation pathways; both types of mechanisms may be inhibited by antioxidants, which can scavenge either radicals or electrophiles. 相似文献
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When rat liver mitochondria are treated with tert-butyl hydroperoxide (TBHP) in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), electron paramagnetic resonance (EPR) signals are detected attributable to spin adducts resulting from the trapping of methyl, tert-butoxyl, and tert-butylperoxyl radicals. The addition of respiratory substrate results in a 3- to 7.5-fold increase in the signal intensity of the DMPO/methyl adduct, no change in the signal intensity of the DMPO/tert-butoxyl adduct, and complete loss of the DMPO/tert-butylperoxyl adduct signal. The magnitude of increase of methyl radical production in the presence of respiratory substrate is related to the respiratory control ratio (RCR) of the mitochondrial preparation. In the presence of antimycin A, which blocks electron flow between cytochromes b and c1, no stimulation of methyl radical production is detected with respiratory substrate. Stimulation of methyl radical production by the addition of respiratory substrate is detected in cytochrome c-depleted mitochondria. A similar increase in methyl radical production is detected when ferrous cytochrome c is treated with TBHP in the presence of DMPO (as compared to when ferricytochrome c is used). These results indicate that TBHP is reduced directly by either cytochrome c1, cytochrome c, or by both of these electron transport chain components in mitochondria undergoing state 4 respiration. 相似文献
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Samuel Rommelaere Virginie Millet Thomas Gensollen Christophe Bourges Jérôme Eeckhoute Nathalie Hennuyer Eric Baugé Lionel Chasson Ivana Cacciatore Bart Staels Giuseppina Pitari Franck Galland Philippe Naquet 《FEBS letters》2013
The membrane-bound Vanin-1 pantetheinase regulates tissue adaptation to stress. We investigated Vnn1 expression and its regulation in liver. Vnn1 is expressed by centrolobular hepatocytes. Using novel tools, we identify a soluble form of Vnn1 in mouse and human serum and show the contribution of a cysteine to its catalytic activity. We show that liver contributes to Vanin-1 secretion in serum and that PPARalpha is a limiting factor in serum Vnn1 production. Functional PPRE sites are identified in the Vnn1 promoter. These results indicate that serum Vnn1 might be a reliable reporter of PPARalpha activity in liver. 相似文献
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Calcium-mediated inhibition of glucuronide production by epinephrine in the perfused rat liver 总被引:2,自引:0,他引:2
S A Belinsky F C Kauffman P M Sokolove T Tsukuda R G Thurman 《The Journal of biological chemistry》1984,259(12):7705-7711
Rates of production of p-nitrophenyl glucuronide by isolated perfused livers from fed or fasted phenobarbital-treated rats were estimated by monitoring the concentration of glucuronide in the effluent perfusate. Infusion of epinephrine decreased the steady state level of p-nitrophenyl glucuronide by about 39% (half-maximal inhibition at approximately 5 microM). This result was unexpected because epinephrine activated glycogenolysis and elevated hepatic UDP-glucuronic acid contents. The effect of epinephrine can be attributed to its interaction with alpha-adrenergic receptors, since the inhibition of glucuronide production by epinephrine was reversed by an alpha-antagonist (phentolamine) but not by a beta-antagonist, propranolol. Since alpha-adrenergic agonists increase the cytosolic free calcium concentration, we investigated the possibility that the decrease in glucuronide production elicited by epinephrine was mediated by calcium. Removal of calcium from the perfusion fluid diminished the inhibition of glucuronide production by epinephrine, while increasing extracellular calcium from 0 to 150 microM restored the inhibition in a dose-dependent manner. In the presence of extracellular calcium, glucuronide production was inhibited by the addition of the calcium ionophore A23187 or angiotensin II, a hormone which increases cytosolic calcium. Concentrations of ionized calcium comparable to physiological intracellular levels (0.1-2 microM) increased microsomal beta-glucuronidase activity by 50 to 100% but had no effect on microsomal glucuronosyl-transferases . These results indicate that activation of hepatic alpha-adrenergic receptors increases cytosolic calcium which stimulates microsomal beta-glucuronidase activity. This decreases net glucuronide formation by the liver. In support of this hypothesis, rates of glucuronide production were unaffected by epinephrine in perfused livers from beta-glucuronidase-deficient C3H/HeJ mice. 相似文献
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1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat. 相似文献
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Biogenesis of mitochondrial proteins. Regulation of production of delta-aminolaevulinate synthase by haemin in embryonic-chick liver. 总被引:2,自引:2,他引:0
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The effect of haemin on the biogenesis of delta-aminolaevulinate synthase (ALA synthase) was investigated in primary cultures of embryonic-chick liver. The activity of the enzyme and the amount of the enzyme detected by 'immune-blotting' were determined in hepatocytes incubated with the porphyrogenic agent allylisopropylacetamide. The results of these studies indicated that the loss in ALA synthase activity in cells incubated in the presence of haemin (10 microM) was roughly proportional to a loss in the immune-reactive mass of the enzyme. Haemin was as effective as cycloheximide in causing depletion of ALA synthase in hepatocytes. We had previously established that haemin blocked the maturation of the precursor of ALA synthase [Ades (1983) Biochem. Biophys. Res. Commun. 110, 42-47]. From results reported in the present paper on analyses of immune-precipitated ALA synthase after pulse-labelling with [35S]methionine in the presence and in the absence of haemin, we determined that the inhibition of processing of pre-ALA synthase in cells by haemin was concentration-dependent. A concentration of 2 microM in the culture medium blocked the processing of pre-ALA synthase by 50% in hepatocytes. We also determined that, after inhibition of its maturation by haemin, pre-ALA synthase turned-over with a half-time of 30 min; on the other hand, mature ALA synthase turned-over with a half-time of 120 min. 相似文献
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Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats. 总被引:2,自引:0,他引:2
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In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO. 相似文献
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B G Miguel M C Calcerrada L Martín R E Catalán A M Martínez 《Prostaglandins & other lipid mediators》2001,65(4):159-166
When isolated rat liver nuclei were treated with platelet-activating factor (PAF), a rapid increase in the mass of diacylglycerol (DAG) occurred. This effect was dose- and time-dependent. The maximum effect was observed after 1 min of 10(-7) M PAF treatment. A concomitant decrease of polyphosphoinositides and phosphatidic acid (PA) levels was observed. PAF-induced DAG accumulation was inhibited by the treatment with WEB 2086 or PCA-4248, specific PAF-receptor antagonists. This result may suggest that PAF exerts its action in the nucleus through specific nuclear PAF binding sites. The findings described herein are due to the activation of phospholipase C, as the results from experiments using U73122, a phospholipase C inhibitor, indicate. These are the first data on the action of 相似文献
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