Adaptive,model-based control by the Open-Loop-Feedback-Optimal (OLFO) controller for the effective fed-batch cultivation of hybridoma cells

Although fed-batch suspension culture of animal cells continues to be of industrial importance for the large scale production of pharmaceutical products, existing control concepts are still insufficient. Changes in cell metabolism during cultivation and between similar cultivations, the complexity of the cell metabolism, and the lack of on-line state variables restrict the transfer of available control strategies established in bioprocess engineering. A process control strategy designed to achieve optimized process control must account for all these difficulties and fit sophisticated requirements toward adaptability and flexibility. The combination of a fed-batch process and an Open-Loop-Feedback-Optimal (OLFO) control provides a new approach for cell culture process control that couples an efficient cultivation concept to a capable process control strategy. The application of an adaptive, model-based OLFO controller to a hybridoma cultivation and experimental results are presented.     

Stability of interleukin 6, soluble interleukin 6 receptor,interleukin 10 and CC16 in human serum

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 degrees C, 20 degrees C, 30 degrees C and 40 degrees C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 degrees C, 20 degrees C and 30 degrees C. Interleukin-6 concentrations significantly declined after 11 days at 40 degrees C. The concentrations of sIL-6R and IL-10 did not change at 4 degrees C but significantly decreased at 20 degrees C (after 21 and 14 days respectively), 30 degrees C and 40 degrees C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at -20 degrees C and -70 degrees C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 degrees C or 20 degrees C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at -20 degrees C for several years, but for IL-10 determinations, storage at -70 degrees C is recommended.     

Structural analysis of the oligosaccharide alditols released from the jelly coat of Rana dalmatina eggs by reductive beta-elimination

A combination of ion-exchange chromatography and high performance liquid chromatography (HPLC) has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of oviducal mucins obtained from the jelly coat of Rana dalmatina. The primary structures of 26 O-glycans were determined by one-dimensional and two-dimensional 1H and 1H13C NMR spectroscopy. As observed for 20 other amphibian species, these carbohydrate chains are highly species-specific. The main typical feature of the species R. dalmatina consists in the presence of the backbone Gal(beta1-3)[Gal(beta1-4)]Gal(beta1-3)GalNAc-ol, previously observed among Ranidae, such as R. temporaria and R. ridibunda. Nevertheless, the nature of carbohydrates present at the periphery of the glycans perfectly differentiates the three species.     

Neuron navigator: a human gene family with homology to unc-53, a cell guidance gene from Caenorhabditis elegans

We have cloned the gene neuron navigator-1 (NAV1), a human homolog of unc-53, a gene involved in axon guidance in Caenorhabditis elegans. Duplications during evolution gave rise to three human homologs located on chromosomes 1q32.1, 11p15.1, and 12q21.1. NAV1 and NAV2 are expressed in the developing brain. NAV1, NAV2, and NAV3 expression is detected in adult heart, kidney, and brain, respectively. NAV1 encodes a protein lacking, in the aminoterminal part, a CH domain present in the other NAV genes. The first exon of NAV1 arose through an ancient internal duplication of sequences that also gave rise to exon 8 of NAV3 and exon 7 of NAV2. A detailed study of the NAV environment on the different chromosomes reveals incomplete micro-syntheny between the three regions. Through analysis of the phylogenetic relationships for three different gene families in the NAV environment, we reconstructed part of the events that formed these regions.     

Heme oxygenase-1 induction may explain the antioxidant profile of pentaerythrityl trinitrate

The organic nitrate pentaerythrityl tetranitrate (PETN) is known to exert long-term antioxidant and antiatherogenic effects by as yet unidentified mechanisms. In cultured endothelial cells derived from human umbilical vein, the active PETN metabolite PETriN (0.01-1 mM) increased heme oxygenase (HO)-1 mRNA and protein levels in a concentration-dependent fashion. HO-1 induction was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of carbon monoxide and bilirubin. Pretreatment with PETriN or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by PETriN were not mimicked by isosorbide dinitrate, another long-acting nitrate. The present study demonstrates that PETriN stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein HO-1 in endothelial cells. Increased HO-1 expression and ensuing formation of cytoprotective bilirubin may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN.     

Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer

Computerized measuring devices are needed to assess canine semen quality objectively both for research and practical purposes. As internal image settings may influence the results considerably, the effect of different technical settings and semen processing on the parameters assessed by the Hamilton-Thorne Ceros 12.1 semen analyzer (HTR Ceros 12.1) was investigated. The frame rate (15, 30 or 60 frames/s) significantly (P<0.05) influenced most of the measured motility characteristics in experiment 1 while no differences in the motility parameters were found using a different sampling duration (0.5 or 1 s, i.e. 30 or 60 frames scanned) in experiment 2. In experiment 3, an increase in sperm velocity (VAP, VSL, VCL), in linearity and in the percentage of motile and rapidly moving spermatozoa was observed with increasing sperm concentrations (25 x 10(6), 50 x 10(6) or 100 x 10(6) ml(-1)). In experiment 4, a clear effect of the diluent used was visible with higher velocity parameters (VAP, VSL, VCL) and higher percentages of motile, progressive and rapid spermatozoa for semen samples diluted in Hepes-TALP or prostatic fluid in comparison with physiological saline or egg-yolk-Tris extender. In experiment 5, significant (P<0.01) and high correlations were found between the conventional dog semen analysis methods and HTR Ceros 12.1 measurements (n=97 semen samples) for the sperm concentration (r=0.91), the motility (r=0.74) and the progressive motility (r=0.84). In experiment 6, the ejaculates from 21 proven, fertile dogs were compared with the ejaculates of a population (N: 11) of young beagles (1.5 years) but no significant differences in HTR Ceros 12.1 measurements were found between the two groups. Based on our results, diluting dog semen samples to 50 x 10(6) ml(-1) with physiological saline solution and scanning 30 frames at a frame rate of 60 frames/s (i.e. a scanning time of 0.5 s), are the set-up parameters proposed to obtain objective and standardized canine semen motility results using the HTR Ceros 12.1.     

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.     

Cellular compartmentation of cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri

The cellular compartmentation of elements was analysed in the Zn hyperaccumulator Arabidopsis halleri (L.) O'Kane & Al-Shehbaz (=Cardaminopsis halleri) using energy-dispersive X-ray microanalysis of frozen-hydrated tissues. Quantitative data were obtained using oxygen as an internal standard in the analyses of vacuoles, whereas a peak/background ratio method was used for quantification of elements in pollen and dehydrated trichomes. Arabidopsis halleri was found to hyperaccumulate not only Zn but also Cd in the shoot biomass. While large concentrations of Zn and Cd were found in the leaves and roots, flowers contained very little. In roots grown hydroponically, Zn and Cd accumulated in the cell wall of the rhizodermis (root epidermis), mainly due to precipitation of Zn/Cd phosphates. In leaves, the trichomes had by far the largest concentrations of Zn and Cd. Inside the trichomes there was a striking sub-cellular compartmentation, with almost all the Zn and Cd being accumulated in a narrow ring in the trichome base. This distribution pattern was very different from that for Ca and P. The epidermal cells other than trichomes were very small and contained lower concentrations of Zn and Cd than mesophyll cells. In particular, the concentrations of Cd and Zn in the mesophyll cells increased markedly in response to increasing Zn and Cd concentrations in the nutrient solution. This indicates that the mesophyll cells in the leaves of A. halleri are the major storage site for Zn and Cd, and play an important role in their hyperaccumulation. Received: 4 April 2000 / Accepted: 16 May 2000     

19F Nuclear Magnetic Resonance as a Tool To Investigate Microbial Degradation of Fluorophenols to Fluorocatechols and Fluoromuconates

A method was developed to study the biodegradation and oxidative biodehalogenation of fluorinated phenols by ¹⁹F nuclear magnetic resonance (NMR). Characterization of the ¹⁹F NMR spectra of metabolite profiles of a series of fluorophenols, converted by purified phenol hydroxylase, catechol 1,2-dioxygenase, and/or by the yeast-like fungus Exophiala jeanselmei, provided possibilities for identification of the ¹⁹F NMR chemical shift values of fluorinated catechol and muconate metabolites. As an example, the ¹⁹F NMR method thus defined was used to characterize the time-dependent metabolite profiles of various halophenols in either cell extracts or in incubations with whole cells of E. jeanselmei. The results obtained for these two systems are similar, except for the level of muconates observed. Altogether, the results of the present study describe a ¹⁹F NMR method which provides an efficient tool for elucidating the metabolic pathways for conversion of fluorine-containing phenols by microorganisms, with special emphasis on possibilities for biodehalogenation and detection of the type of fluorocatechols and fluoromuconates involved. In addition, the method provides possibilities for studying metabolic pathways in vivo in whole cells.