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The IgLONs are a family of glycosyl phosphatidyl inositollinked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell-cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU-1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU-1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU-1 adhered to a recombinant CEPU-1-Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU-1-Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU-1-Fc, LAMP-Fc and OBCAM-Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON-Fc substrate and overexpression of CEPU-1-GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON-Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non-transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell-cell adhesion and recognition.

Recombinant Fc chimeric proteins are useful tools for studying protein function, including the analysis of molecular interactions by techniques such as expression cloning. Here we describe a method we have used to express the IgLON family proteins, CEPU1 and OBCAM, as recombinant Fc chimeric proteins in stably transfected mouse J558L myeloma cells. The use of this cell line provided the opportunity to maximize protein production, as it secretes antibodies in large quantities and can be grown to high density in small volumes of culture medium. Isolation of recombinant OBCAMFc from the adherent COS7 cell line suggested a minimum level of expression of 0.07 mg OBCAMFc/100 mL culture medium, while the J558L cell line expressed OBCAMFc at approximately 11.4 mg/100 mL culture medium. Purification of IgLON-Fc expressed by J558L cells was simpler than purification from COS7 cells because of the lower volume of culture medium generated. Furthermore, contamination of J558L expressed IgLONFc with bovine IgG from the culture medium was negligible. The method presented, which utilizes a commercially available small-scale bioreactor, provides the nonspecialist protein expression laboratory with the means to produce recombinant proteins quickly and easily in milligram quantities.

In a screen for myosin-like proteins in embryonic chicken brain, we have identified a novel nuclear protein structurally related to hnRNP-U (heterogeneous nuclear ribonuclear protein U). We have called this protein chURP, for chicken U-related protein. In this screen, chURP was immunoreactive with two myosin antibodies and, in common with the unconventional myosins, bound calmodulin in vitro in both the presence and absence of calcium ions. Determination of 757 amino acids of the chURP sequence revealed that it shares 41% amino acid identity with human and rat hnRNP-U, although chURP and hnRNP-U appear not to be orthologous proteins. ChURP is ubiquitously expressed in the nuclei of all chick tissues and, as one of a growing number of calmodulin-binding proteins to be identified in the nucleus, further highlights the potential of calmodulin as a regulator of nuclear metabolism.Keywords: hnRNP particles; nuclear matrix; calmodulin; hnRNP-U.Protein-coding genes in eukaryotes are transcribed by RNA polymerase II to form nascent transcripts which, because of their wide size distribution and subcellular localization, are known as heterogeneous nuclear (hn)RNAs. As soon as hnRNAs begin to emerge from their genes, they associate cotranscriptionally with a variety of proteins to become heterogeneous nuclear ribonucleoprotein (hnRNP) complexes [1,2]. The macromolecular composition of hnRNP complexes is diverse and includes spliceosomes, which are themselves ribonucleoprotein complexes consisting of a variety of small nuclear (sn)RNAs and associated proteins in the form of snRNPs [3,4]. Those proteins associated with hnRNAs that are not stable components of RNP complexes such as spliceosomes are generally called hnRNP proteins [1,5]. Approximately 20 major hnRNP proteins, designated hnRNP-A1 through -U in order of ascending molecular weight, are typically identified in hnRNP complexes immunopurified from various sources [1,6±10]. The cDNAs for many of these proteins have now been cloned although their functions are not completely understood [1,5,11]. For example, various hnRNP proteins have been demonstrated to possess RNA annealing activity which may be important in both cis and trans interactions during pre-mRNA splicing [12±18], while the same proteins may also be involved in regulating the export of mature mRNAs from the nucleus to the cytoplasm [19±27]. HnRNP proteins therefore appear to have complex functions.Consistent with their proposed roles in vivo, hnRNP proteins have been demonstrated to bind RNA in vitro [1,5,10]. Three distinct RNA-binding domains (RBDs), known as the RNP-CS, KH and RGG box motifs, are represented in the hnRNP proteins, sometimes in combination within one particular polypeptide [28]. For example, hnRNP-A1 contains two RNP-CS motifs and one RGG box. The RNA-binding activity of recombinant hnRNP-U has been mapped to a 26-amino-acid RGG box motif in a C-terminal 112 amino acid domain termed U-gly, as it is rich in glycine residues [29]. Interestingly, hnRNP-U has also been independently charact...

The regulation of cell‐based medicinal products in the EU is governed by legislation for medicinal products in general plus dedicated regulations for advanced therapy medicinal products (ATMPs). In addition, the procurement and use of the tissues and cells used to manufacture these products are regulated by additional legislation. This presentation will focus on how a regulatory strategy for bringing a cell‐based ATMP under development into clinical trials and ultimately to marketing authorisation should be formulated around all relevant legislation and the guidelines used to implement it. A regulatory strategy should address the quality, safety and efficacy aspects of a medicinal product intended for human use, through defined strategies around CMC (chemistry, manufacturing and controls), nonclinical studies and clinical trials. In addition, regulatory routes to clinical trials and market authorisation that are appropriate to the ATMP under development should also be considered from an early stage, for example including orphan designation, paediatric investigation plans and accelerated approval schemes. Building a regulatory and product development strategy around these elements for a cell‐based ATMP will be outlined.

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