Effects of reactive oxygen metabolites on erythropoietin production in renal carcinoma cells

The present studies were undertaken to determine the effects of reactive oxygen metabolites on erythropoietin (Ep) biosynthesis in Ep-producing renal carcinoma (RC) cells using a sensitive radioimmunoassay for Ep. Xanthine (10-5M) and increasing concentrations of xanthine oxidase (8 x 10(-7) to 5 x 10(-4) units/ml) produced a significant dose-related increase in Ep production at a concentration of greater than or equal to 4 x 10(-6) units/ml, whereas xanthine alone had no effect. Catalase, a scavenger of hydrogen peroxide (H2O2), in concentrations of 50 to 500 micrograms/ml produced a significant inhibition of the increase in Ep production induced by xanthine-xanthine oxidase; while no effect was seen on basal levels of Ep production and the growth of RC cells. Glucose oxidase (greater than or equal to 0.032 mU/ml), a direct H2O2 generator, and exogenous H2O2 (greater than or equal to 4 x 10(-6)M) added to the incubation mixture, caused a significant enhancement of Ep production in a dose-dependent manner. Xanthine-xanthine oxidase, glucose oxidase, and H2O2 in the above concentrations did not produce significant cytotoxicity (51Cr release or trypan blue dye exclusion). The present data suggests that H2O2, a reactive oxygen metabolite may play a significant role in Ep production.     

Continuous production of erythropoietin with immobilized animal cells

Summary Erythropoietin, a glycoprotein that is a physiological stimulator of erythrocyte production, was produced continuously for more than 32 days by three kinds of anchorage-dependent animal cells immobilized in alginate gel particles. Gelation caused by divalent cations added to an alginate solution containing cells resulted in the formation of clearly vacant spaces (referred to here as channels) with prolate ellipsoidal shapes inside the gel particles. Each channel originated from a cell and extended towards the center of the gel particle. The animal cells grew well three-dimensionally in the channels but proliferated little outside the channels. Most of the channels had been filled with cells 2 weeks after immobilization. The cell concentration in the gel particles reached more than 1×10⁷ cells/g gel. The alginate immobilization method was useful for high-concentration cultivation of the anchorage-dependent cells.     

Effect of cis-diamminedichloroplatinum on erythropoietin production and hematopoietic progenitor cells

Studies were conducted to determine whether the progressive development of anemia associated with the antineoplastic drug cis-diamminedichloroplatinum (cDDP) was the consequence of decreased erythropoietin (Epo) production due to cDDP-induced nephrotoxicity or selective inhibition of erythroid progenitor cells. Five days after a single intraperitoneal injection of cDDP, hypoxia-induced Epo production was not decreased in mice and was increased significantly in rats in spite of severe multifocal tubular necrosis. In both species, colony-forming units-granulocyte macrophage (CFU-gm) and colony-forming units-erythroid (CFU-e) were reduced significantly, with a greater decrease in CFU-e. Studies of an anemic patient receiving cDDP also showed elevated Epo and decreased CFU-gm and CFU-e. In vitro exposure of mouse and human bone marrow to cDDP caused a dose-dependent inhibition of CFU-gm and CFU-e in both species, with human CFU-e showing greatest sensitivity. The results indicate that the primary hematologic toxicity of cDDP is directed at the hematopoietic stem cell compartment.     

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.     

Sensitivity of yeast cells to reactive oxygen species generated in the extracellular space

Even when cytoplasmic scavenging activities are plentiful, yeast cells (S. cerevisiae) remain particularly sensitive towards reactive oxygen species generated in the extracellular space (either by the xanthine/xanthine oxidase reaction or by the redox cycling of menadione). A sharp reduction of the extent of cellular alterations when SOD and/or catalase were supplemented in the incubation buffer, points to a contribution of both O-.2 and H2O2 in the toxic process. Although oxygen metabolites as well as t-butylhydroperoxide (tBH), a highly toxic organic peroxide, may be directly responsible for cellular damage, their toxicity is largely reduced in the presence of Desferal. A role of metal ions in potentiating the toxicity points to the involvement of OH. radicals, actually produced in the medium. With tBH, metal cations would be rather active in promoting peroxidative chain reactions. In the case of an extracellular oxidative attack, it may be foreseen that the plasma membrane will form a preferential target. An increased permeability of the plasma membrane towards ionized molecules and uncharged polycarboxylic acids is indeed observed after an oxidative treatment. The loss of selective permeability is, as a rule, correlated with a drop in viability. Early alterations, disrupting the functional organization of the plasma membrane have been sought. The permease involved in the active transport of purine(s) has appeared to be an appropriate marker for checking its functional integrity. This transport function appears to be very sensitive to damage induced by O-.2 generators, particularly under conditions in which the resulting lethality is still kept low and in which the energization of active transport processes remains unimpaired.     

Continuous production of human erythropoietin by immobilized recombinant L-929 cells

Recombinant L-929 cells transfected with the human erythropoietin (EPO) gene were immobilized in a macroporous cellulosic support and its derivatives in which charged groups or cell attachment factors were introduced. The immobilized cells were cultured in serum-containing and serum-free media. Comparable production of EPO was observed even in the serum-free medium when a support modified by polyethyleneimine was used for immobilization. The cells immobilized on the supports were cultured in fluidized-bed and inner-loop type air-lift bioreactors for continuous production of EPO. A high cell density of more than 2 × 10⁷ cells/cm³-support and high EPO productivity were achieved and maintained for 50 d through the use of the inner-loop type air-lift bioreactor. The productivity was 13.4-fold higher than that of conventional static cultures in petri-dishes.     

Calcium response to erythropoietin in erythroleukemia cells

Erythroleukemic mouse spleen cells were analyzed by flow cytometry at three fluorescence wavelengths, two emissions for the calcium indicator indo 1 and immunofluorescence with an antiserum directed against murine leukemia retrovirus antigen. The antigen-positive subpopulation of cells responded to EPO by transiently changing indo 1 fluorescence emissions, indicating a change in cytoplasmic calcium concentration. EGTA blocked the EPO response, suggesting that Ca2+ influx may have followed EPO exposure in these cells. Antigen-negative cells showed no Ca2+ response to EPO. Infected mice all contained antigen-positive spleen cells, but, as measured by changes in indo 1 fluorescence, these subpopulations varied in their ability to respond to EPO.     

The influence of phenobarbital on cytochromes and reactive oxygen species in erythropoietin producing HepG2 cells

Light absorption photometry of HepG2 cells treated with phenobarbital for enhancing the content of cytochrome P-450 and the synthesis of erythropoietin revealed an influence on all cytochromes detectable in the wavelength range between 400 and 620 nm. No correlation was found between specific changes of cytochrome P-450 absorption and increased EPO synthesis as proposed earlier by Fandrey et al. (Life Sci. (1990) 47, 127–134). In the present study, however, the increased erythropoietin synthesis could be related to a decreased intracellular hydroxyl radical level described as crucial for the oxygen regulated gene expression (Kietzmann et al., Biochem. J. (1998) 335, 425–432; Porwol et al., Eur. J. Biochem. (1998) 256, 16–23).     

Sensitivity of methanogenic bacteria from paddy soil to oxygen and desiccation

Abstract The effects of combinations of desiccation and exposure to O₂ were studied in pure cultures of Methanosarcina barkeri strain Fusaro and in a new Methanosarcina strain and a new Methanobacterium strain which were both isolated from dry oxic paddy soil. Incubation of bacterial suspensions under air for 200 min resulted in a decreased potential to produce CH₄, but not in a decreased viability. The inhibitory effect of O₂ slightly increased with increased salt concentration. Desiccation of bacterial suspensions under N₂ resulted in reduction of viability to 10% and of potential CH₄ production to 0.6%. Desiccation of bacterial suspensions under air resulted in a larger decrease of both viability (0.5%) and potential CH₄ production (0.03%). This decrease was smaller at rapid compared to slow desiccation. Survival and potential CH₄ production were further inhibited when the suspension was dried in the presence of sand grains or glass beads coated with FeS or FeNH₄PO₄. However, survival and potential CH₄ production increased dramatically in the presence of pyrite (FeS₂) grains. Then, as much as 10% of the initial methanogenic population survived oxic desiccation. This relatively good resistance is in agreement with observations that methanogens in rice fields survive the periods when the paddy soil is dry and oxic.     

Current state and perspectives on erythropoietin production

Erythropoietin is a major regulator of erythropoiesis which maintains the body's red blood cell mass and tissue oxygenation at an optimum level. Recombinant human erythropoietin (rhEPO), which is a widely used therapeutic agent for the treatment of anemia and which represents one of the largest biopharmaceuticals markets, is produced from recombinant Chinese hamster ovary cells. rhEPO is a glycoprotein with complex glycan structure, which is responsible for its therapeutic efficacy, including the in vivo activity and half-life. In order to obtain an optimal and consistent glycoform profile of rhEPO and concurrently maintain a high production yield, various approaches in drug development and cell culture technology have been attempted. Recent advances in rhEPO production are classified into three types: the development of improved rhEPO molecules by protein engineering; improvement of production host cells by genetic engineering; and culture condition optimization by fine control of the production mode/system, process parameters, and culture media. In this review, we focus on rhEPO production strategies as they have progressed thus far. Furthermore, the current status of the market and outlook on rhEPO and its derivatives are discussed.     

Sensitivity of mean annual primary production to precipitation

In many terrestrial ecosystems, variation in aboveground net primary production (ANPP) is positively correlated with variation in interannual precipitation. Global climate change will alter both the mean and the variance of annual precipitation, but the relative impact of these changes in precipitation on mean ANPP remains uncertain. At any given site, the slope of the precipitation‐ANPP relationship determines the sensitivity of mean ANPP to changes in mean precipitation, whereas the curvature of the precipitation‐ANPP relationship determines the sensitivity of ANPP to changes in precipitation variability. We used 58 existing long‐term data sets to characterize precipitation‐ANPP relationships in terrestrial ecosystems and to quantify the sensitivity of mean ANPP to the mean and variance of annual precipitation. We found that most study sites have a nonlinear, saturating relationship between precipitation and ANPP, but these nonlinearities were not strong. As a result of these weak nonlinearities, ANPP was nearly 40 times more sensitive to precipitation mean than variance. A 1% increase in mean precipitation caused a ?0.2% to 1.8% change in mean ANPP, with a 0.64% increase on average. Sensitivities to precipitation mean peaked at sites with a mean annual precipitation near 500 mm. Changes in species composition and increased intra‐annual precipitation variability could lead to larger ANPP responses to altered precipitation regimes than predicted by our analysis.     

Sensitivity of chondrocytes of growing cartilage to reactive oxygen species

Vascular invasion of calcified cartilage, during endochondral ossification, is initiated and sustained by invasive cells (endothelial cells and macrophages) which degrade the tissue by releasing lytic enzymes. Concurrently, reactive oxygen species (ROS) are also released by these cells and we hypothesize that ROS also contribute to the degradation of the tissue. As a preliminary approach to this problem, the antioxidant activities and the effect of ROS on hypertrophic cartilage and chondrocytes (HCs) were investigated. Compared to resting or articular chondrocytes, HCs exhibited higher catalase but lower SOD specific activities and lower PHGPx concentration, thus revealing a defence activity specific against H₂O₂. Moreover, dose-dependent depletion of ATP occurred after few minutes of exposure to ROS, and a long-term treatment (16 h incubation with ROS) promoted the release of LDH activity and a significant variation of the poly- to mono-unsaturated fatty acid ratio. Finally, the incubation of HCs with low ROS doses induced the release of sedimentable alkaline phosphatase activity (matrix vesicles). How the obtained results fit the in vivo occurring events is discussed.