Simple assay of serum copper fractions by ultrafiltration and flameless atomic absorption

The authors present a simple and rapid method for assaying ultrafilterable copper (Cu UF) and albumin-bound copper (Cu ALB). It is based on the ultrafiltration of serum in the presence of ethylenediaminetetraacetic acid (EDTA), used to prevent adsorption on the membranes. EDTA at 0.4 g/L has no effect on the equilibrium of serum copper vectors and enables Cu UF to be assayed by flameless atomic absorption. EDTA at 2 g/L, is used to assay total exchangeable copper (CU EXC) (ultrafilterable+albumin-bound). The evaluation criteria of the method are furnished, as are the normal values in healthy subjects: 14.6 μg/L for Cu UF and 87 μg/L for Cu EXC. Finally, the usefulness of the methods described here for the diagnosis of Wilson's and Menkes' disease was demonstrated.     

Monitoring of temperature effects on animal cell metabolism in a packed bed process

Animal cell (Chinese Hamster Ovary) concentration was determined on-line in a packed bed process using dielectric spectroscopy. This enabled the evaluation of the effect of temperature on specific metabolic rates during 3 months of continuous culture. The effect of low cultivation temperature on cell growth and metabolism was monitored, and the data were used for process development. At 37 degrees C cells grew exponentially with a specific growth rate of 0.038 d-1 and specific glucose uptake and lactate production rates increased continually. Reduction of the temperature to 33.5 degrees C resulted in a lowering of these metabolic rates while having no effect on cell proliferation. Subsequent reduction of the temperature to 32 degrees C resulted in stabilization of the cell concentration at a high density (3.6 x 10(7) cell per mL of packed bed). In addition, the specific production rate of the protein of interest increased by a factor of 6 compared to the value at 37 degrees C. During the stationary phase at 32 degrees C, all other specific metabolic rates could be controlled to low and constant levels.     

Serum T-kininogen levels increase two to four months before death.

We have reported an accumulation of T-kininogen mRNA in the liver of aging Sprague-Dawley rats. T-kininogen is a cysteine proteinase inhibitor. Since a disruption of the intracellular protein degradation machinery is known to occur during senescence, we wished to further define the role of this protein in the aging process. As a first step, we have measured T-kininogen levels both in serum and within the liver. We have found that serum protein levels are indeed augmented during senescence, although not as dramatically as the mRNA (2.5-fold versus 8.3-fold). Immunocytochemistry, as well as Western blot analysis suggests that this is due to the presence of T-kininogen within hepatic cells in aged rats. Life-long dietary restriction, a known age-prolonging treatment, decreases the overexpression of the protein in 24-month-old rats. Later, diet-restricted animals still show an increased expression from the gene, the effect being delayed but not abolished by dietary manipulation. Interestingly, a longitudinal study indicated the existence of a positive correlation between the time of increase of serum T-kininogen and the time of death of the animal. Serum T-kininogen was found to increase 2.5-4 months before death.     

Packed-bed bioreactors for mammalian cell culture: bioprocess and biomedical applications

This article describes the development history of packed-bed bioreactors (PBRs) used for the culture of mammalian cells. It further reviews the current applications of PBRs and discusses the steps forward in the development of these systems for bioprocess and biomedical applications. The latest generation of PBRs used in bioprocess applications achieve very high cell densities (>10(8) cells ml(-1)) leading to outstandingly high volumetric productivity. However, a major bottleneck of such PBRs is their relatively small volume. The current maximal volume appears to be in the range of 10 to 30 l. A scale-up of more than 10-fold would be necessary for these PBRs to be used in production processes. In biomedical applications, PBRs have proved themselves as compact bioartificial organs, but their metabolic activity declines frequently within 1 to 2 weeks of operation. A main challenge in this field is to develop cell lines that grow consistently to high cell density in vitro and maintain a stable phenotype for a minimum of 1 to 2 months. Achieving this will greatly enhance the usefulness of PBR technology in clinical practice.     

Implementing environmental labelling of food products in France

Consumers increasingly demand information about the environmental impacts of their food. The French government is in the process of introducing environmental labelling for all food products. A scientific council was set up, and its main conclusions are presented in this article, through six questions: What environmental issues should be considered? What objective should be targeted? What data are needed, and for whom? What methods for assessing environmental impacts? Which environmental scores should be chosen? What label format should be proposed? By answering these questions and considering the context, the available data, the proposed methods and adjustments, and the knowledge of consumer perception of formats, the scientific council considers that a labelling scheme is feasible and relevant.

     

Use of glucose consumption rate (GCR) as a tool to monitor and control animal cell production processes in packed-bed bioreactors

For animal cell cultures growing in packed-bed bioreactors where cell number cannot be determined directly, there is a clear need to use indirect methods that are not based on cell counts in order to monitor and control the process. One option is to use the glucose consumption rate (GCR) of the culture as an indirect measure to monitor the process in bioreactors. This study was done on a packed-bed bioreactor process using recombinant CHO cells cultured on Fibra-Cel disk carriers in perfusion mode at high cell densities. A key step in the process is the switch of the process from the cell growth phase to the production phase triggered by a reduction of the temperature. In this system, we have used a GCR value of 300 g of glucose per kilogram of disks per day as a criterion for the switch. This paper will present results obtained in routine operations for the monitoring and control of an industrial process at pilot-scale. The process operated with this GCR-based strategy yielded consistent, reproducible process performance across numerous bioreactor runs performed on multiple production sites.     

A new method for on-line measurement of the volumetric oxygen uptake rate in membrane aerated animal cell cultures

Oxygen is a key substrate in animal cell metabolism and its consumption is thus a parameter of great interest for bioprocess monitoring and control. A system for measuring it based on an oxygen balance on the liquid phase was developed. The use of a gas-permeable membrane offered the possibility to provide the required quantity of oxygen into the culture, while avoiding problems of foaming or shear stress generally linked to sparging. This aeration system allowed moreover to keep a known and constant k(L)a value through cultures up to 400 h. Oxygen uptake rate (OUR) was measured on-line with a very good accuracy of +/-5%, and the specific OUR for a CHO cell line was determined during batch (growth phase) and continuous culture as, respectively, equal to 2. 85x10(-13) and 2.54x10(-13) mol O(2) cell(-1) h(-1). It was also shown that OUR continuous monitoring gives actually more information about the metabolic state of the culture than the cell concentration itself, especially during transition phases like the end of the growth phase in a batch culture.     

The effect of an admixture of sodium hydrogen phosphate or heparin-coating to poly(D,L)lactide--results of an animal study.

The study was aimed at investigating the effect of an admixture of sodium hydrogen phosphate (NaP) on the pH value around degrading poly(D,L)lactide (PDLLA) and the possible improvement of PDLLA biocompatibility by coating its surface with heparin. PDLLA +/- NaP was injection-molded to form rods (20 x 3 x 2 (mm)) and cubes (3 x 2 x 2 (mm)). Half of the pure PDLLA samples were surface-coated using heparin. One rod and cube each of PDLLA, PDLLA + NaP and PDLLA/Hep were implanted into the dorsal muscles of 42 rats. From the 2nd to 52nd week after operation, pH measurements were performed in the environment around the implants. The samples were then harvested for histological and mechanical analyses. No significant decrease in pH-values was observed in the tissue around the implants. Pure PDLLA and PDLLA/Hep samples were macroscopically resorbed after 52 weeks, while the degradation of PDLLA + NaP was still in progress. Approximately 80% of the initial bending strength of PDLLA or PDLLA/Hep rods was present after six weeks, while the bending strength of PDLLA + Nap was reduced to 50% after 4 weeks. Heparin-coating of PDLLA did not improve its biocompatibility but did increase its resorption. While no significant effect of NaP on pH value was found, its admixture did reduce the mechanical characteristics of the implants.     

Hyperactive S6K1 mediates oxidative stress and endothelial dysfunction in aging: inhibition by resveratrol

Mammalian target of rapamycin (mTOR)/S6K1 signalling emerges as a critical regulator of aging. Yet, a role of mTOR/S6K1 in aging-associated vascular endothelial dysfunction remains unknown. In this study, we investigated the role of S6K1 in aging-associated endothelial dysfunction and effects of the polyphenol resveratrol on S6K1 in aging endothelial cells. We show here that senescent endothelial cells displayed higher S6K1 activity, increased superoxide production and decreased bioactive nitric oxide (NO) levels than young endothelial cells, which is contributed by eNOS uncoupling. Silencing S6K1 in senescent cells reduced superoxide generation and enhanced NO production. Conversely, over-expression of a constitutively active S6K1 mutant in young endothelial cells mimicked endothelial dysfunction of the senescent cells through eNOS uncoupling and induced premature cellular senescence. Like the mTOR/S6K1 inhibitor rapamycin, resveratrol inhibited S6K1 signalling, resulting in decreased superoxide generation and enhanced NO levels in the senescent cells. Consistent with the data from cultured cells, an enhanced S6K1 activity, increased superoxide generation, and decreased bioactive NO levels associated with eNOS uncoupling were also detected in aortas of old WKY rats (aged 20-24 months) as compared to the young animals (1-3 months). Treatment of aortas of old rats with resveratrol or rapamycin inhibited S6K1 activity, oxidative stress, and improved endothelial NO production. Our data demonstrate a causal role of the hyperactive S6K1 in eNOS uncoupling leading to endothelial dysfunction and vascular aging. Resveratrol improves endothelial function in aging, at least in part, through inhibition of S6K1. Targeting S6K1 may thus represent a novel therapeutic approach for aging-associated vascular disease.