Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Characterization of apoptosis in PER.C6® batch and perfusion cultures
    Mercier, S.M. ; Diepenbroek, B. ; Martens, D.E. ; Wijffels, R.H. ; Streefland, M. - \ 2015
    Biotechnology and Bioengineering 112 (2015)3. - ISSN 0006-3592 - p. 569 - 578.
    hamster ovary cells - high-level expression - flow-cytometry - line per.c6 - death - adenovirus - dna - vaccine - gene - manufacture
    Preventing or delaying cell death is a challenge in mammalian cell cultures for the development and optimization of production processes for biopharmaceuticals. Cell cultures need to be maintained highly viable for extended times in order to reach maximum production yields. Moreover, programmed cell death through apoptosis is often believed to occur without being detected by classical viability measurements. In this study, we characterized cell death in PER.C6® batch and perfusion cultures using three flow cytometry techniques measuring different steps of the apoptosis cascade: DNA fragmentation, caspases activation and phosphatidylserine externalization. We showed that apoptosis is the main pathway of PER.C6® cell death in batch cultures after depletion of main carbon sources. In high cell density perfusion cultures fed at a constant specific perfusion rate, both high viability and very limited apoptosis were observed. When extending this perfusion process far beyond standard operations, cultures were exposed to suboptimal process conditions, which resulted in an increase of apoptotic cell death. Moreover, we showed that the reference viability measurement using trypan blue exclusion properly assesses the level of cell death in PER.C6® cultures. This study is a first step in understanding the mechanisms of PER.C6® cell death, which will be helpful to support applications of the cell line.
    Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis
    Marissen, W.E. ; Kramer, R.A. ; Rice, A. ; Weldon, W.C. ; Niezgoda, M. ; Faber, M. ; Slootstra, J.W. ; Meloen, R.H. ; Clijsters-van der Horst, M. ; Visser, T.J. ; Jongeneelen, M. ; Thijsse, S. ; Throsby, M. ; Kruif, J. de; Rupprecht, C.E. ; Dietzschold, B. ; Goudsmit, J. ; Bakker, A.B.H. - \ 2005
    Journal of Virology 79 (2005)8. - ISSN 0022-538X - p. 4672 - 4678.
    high-level expression - postexposure prophylaxis - glycoprotein - pathogenicity - determinant - virulence - mice - igg
    Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.
    Pentalenene Synthase: Analysis of Active Site Residues by Site-Directed Mutagenesis
    Seemann, M. ; Zhai, G. ; Kraker, J.W. de; Paschall, C.M. ; Christianson, D.W. ; Cane, D.E. - \ 2002
    Journal of the American Chemical Society 124 (2002)26. - ISSN 0002-7863 - p. 7681 - 7689.
    farnesyl-diphosphate synthase - high-level expression - pre-steady-state - crystal-structure - aristolochene synthase - trichodiene synthase - 5-epi-aristolochene synthase - enzymatic cyclization - sterol biosynthesis - molecular-cloning
    Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in kcat and kcat/Km. By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg2+-binding aspartate-rich domain, 80DDLFD, resulted in reduction in the kcat/Km for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a kcat/Km which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product ß-caryophyllene (9) (9%). Finally, the F77Y mutant had a kcat/Km which was reduced by 20-fold compared to that of the wild-type synthase.
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