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contributed equally to this work NRIF (neurotrophin receptor interacting factor) is a ubiquitously expressed zinc finger protein of the Krü ppel family which interacts with the neurotrophin receptor p75 NTR . The interaction was first detected in yeast and then biochemically confirmed using recombinant GST-NRIF fusions and p75 NTR expressed by eukaryotic cells. Transgenic mice carrying a deletion in the exon encoding the p75 NTRbinding domain of NRIF display a phenotype which is strongly dependent upon genetic background. While at the F 2 generation there is only limited (20%) embryonic lethality, in a congenic BL6 strain nrif -/-mice cannot survive beyond E12, but are viable and healthy to adulthood in the Sv129 background. The involvement of NRIF in p75 NTR /NGF-mediated developmental cell death was examined in the mouse embryonic neural retina. Disruption of the nrif gene leads to a reduction in cell death which is quantitatively indistinguishable from that observed in p75 NTR-/-and ngf -/-mice. These results indicate that NRIF is an intracellular p75 NTRbinding protein transducing cell death signals during development.

Cholinergic neurons innervating cortical structures are among the most affected neuronal populations in Alzheimer's disease. In rodents, they express high levels of the neurotrophin receptor p75NTR. We have analyzed cholinergic septohippocampal neurons of the medial septal nucleus in p75exonIII (partial p75NTR knock-out) and p75exonIV (complete p75NTR knock-out) mice, in their original genetic background and in congenic strains. At postnatal day 15, the p75exonIII mutation leads to a moderate increase (+13%) in these neurons among littermates only after back-crossing in a C57BL/6 background. In contrast, the null p75exonIV mutation, which prevents expression of both the full-length and the shorter p75NTR isoforms, results in a 28% neuronal increase, independent of genetic background. The incomplete nature of the p75NTR mutation used previously, coupled with difficulties in delineating the mouse medial septum and the impact of the genetic background on cell numbers, all contribute to explain previous difficulties in establishing the role of p75NTR in regulating cholinergic neuron numbers in the mouse forebrain.

IntroductionSince the early 1990s, recombinant human clotting factor VIII (rhFVIII) produced in hamster cells has been available for haemophilia A treatment. However, the post-translational modifications of these proteins are not identical to those of native human FVIII, which may lead to immunogenic reactions and the development of inhibitors against rhFVIII. For the first time, rhFVIII produced in a human host cell line is available.AimWe describe here the establishment of the first human production cell line for rhFVIII and the manufacturing process of this novel product.Methods and resultsA human cell line expressing rhFVIII was derived from human embryonic kidney (HEK) 293 F cells transfected with an FVIII expression plasmid. No virus or virus-like particles could be detected following extensive testing. The stringently controlled production process is completely free from added materials of animal or human origin. Multistep purification employing a combination of filtration and chromatography steps ensures the efficient removal of impurities. Solvent/detergent treatment and a 20 nm pore size nanofiltration step, used for the first time in rhFVIII manufacturing, efficiently eliminate any hypothetically present viruses. In contrast to hamster cell-derived products, this rhFVIII product does not contain hamster-like epitopes, which might be expected to be immunogenic.ConclusionsHEK 293 F cells, whose parental cell line HEK 293 has been used by researchers for decades, are a suitable production cell line for rhFVIII and will help avoid immunogenic epitopes. A modern manufacturing process has been developed to ensure the highest level of purity and pathogen safety.

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The Dictyostelium protein AmpA (adhesion modulation protein A) is encoded by the gene originally identified by the D11 cDNA clone. AmpA contains repeated domains homologous to a variety of proteins that influence cell adhesion. The protein accumulates during development, reaching a maximal level at the finger stage. Much of the AmpA protein is found extracellularly during development, and in culminants, AmpA is found in association with anterior-like cells. Characterization of an ampA- strain generated by gene replacement reveals a significant increase in cell-cell clumping when cells are starved in nonnutrient buffer suspensions. Developing ampA- cells are also more adhesive to the underlying substrate and are delayed in developmental progression, with the severity of the delay increasing as cells are grown in the presence of bacteria or on tissue culture dishes rather than in suspension culture. Reintroduction of the ampA gene rescues the developmental defects of ampA- cells; however, expression of additional copies of the gene in wild-type cells results in more severe developmental delays and decreased clumping in suspension culture. We propose that the AmpA protein functions as an anti-adhesive to limit cell-cell and cell-substrate adhesion during development and thus facilitates cell migration during morphogenesis.

Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive ParkinsonÕs disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit a4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The Cterminal IBR-RING domain of parkin and the C-terminal part of a4 were essential for the interaction. Biochemical studies revealed that a4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the Nterminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.