Bravo/Nr-CAM Is Closely Related to the Cell Adhesion Molecules L1 and Ng-CAM and Has a Similar Heterodimer Structure

Diverse cell-surface molecules of the nervous system play an important role in specifying cell interactions during development. Using a method designed to generate mAbs against neural surface molecules of defined molecular weight, we have previously reported on the surface protein, Bravo, found in the developing avian retinotectal system. Bravo is immunologically detected on developing optic fibers in the retina, but absent from distal regions of the same fibers in the tectum. We have isolated cDNA clones encompassing the entire coding region of Bravo, including clones containing five alternative sequences of cDNA. These putative alternatively spliced sequences encode stretches of polypeptide ranging in length from 10-93 amino acids and are predicted to be both extra- and intracellular. The deduced primary structure of Bravo reveals that, like the cell adhesion molecules (CAMs) chicken Ng- CAM and mouse L1, Bravo is composed of six Ig-like domains, five fibronectin type III repeats, a transmembrane domain, and a short cytoplasmic region. Recently, the cDNA sequence of a related molecule, Nr-CAM, was reported and its possible identity with Bravo discussed (Grumet, M., V. Mauro, M. P. Burgoon, G. E. Edelman, and B. A. Cunningham. 1991. J. Cell Biol. 113:1399-1412). Here we confirm this identity and moreover show that Bravo is found on Muller glial processes and end-feet in the developing retina. In contrast to the single polypeptide chain structure of Nr-CAM reported previously, we show that Bravo has a heterodimer structure composed of an alpha chain of M(r) 140/130 and a beta chain of 60-80 kD. As with L1 and Ng-CAM, the two chains of Bravo are generated from an intact polypeptide by cleavage at identical locations and conserved sites within all three molecules (Ser-Arg/Lys-Arg). The similar domain composition and heterodimer structure, as well as the 40% amino acid sequence identity of these molecules, defines them as an evolutionarily related subgroup of CAMs. The relationship of Bravo to molecules known to be involved in cell adhesion and process outgrowth, combined with its pattern of expression and numerous potential isoforms, suggests a complex role for this molecule in cell interactions during neural development

Uridine-Conjugated Ferrocene DNA Oligonucleotides: Unexpected Cyclization Reaction of the Uridine Base

The study of energy and electron-transfer processes through DNA duplexes and the development of DNA hybridization probes and electrochemical sensors have resulted in the incorporation of numerous transition-metal complexes into DNA oliognucleotides. These include ruthenium, osmium, iron, rhodium, and copper complexes. Ferrocene (Fc) and its derivatives are attractive electrochemical probes because of their stability and convenient synthetic chemistry. Fc-containing DNA oligonucleotides have been prepared by attaching ferrocenyl moieties to the 5‘ termini through either solid-phase synthesis using phosphoramidites or by reacting suitable ferrocenyl derivatives with end-functionalized oligonucleotides

Bravo, a novel immunoglobulin superfamily member in the developing avian nervous system, is identified using a new method

Cell-surface molecules play an essential role in guiding axons to their targets. We have developed a method to generate monoclonal antibodies (MAbs) which recognize cell-surface molecules of defined molecular weight that are expressed during the development of the chicken retinotectal system. The antigen distribution on optic fibers recognized by one of these MAbs, Bravo, is restricted to retinal ganglion cell axons in the retina, and absent from these same axons in the tectum. The complete derived sequence of Bravo including four putative alternatively spliced regions has been obtained. It reveals a close relationship to the neural members of the immunoglobulin superfamily, with closest relationship to chicken Ng-CAM and mouse L1. Like Ng-CAM and L1, Bravo contains six immunoglobulin domains, five fibronectin type III repeats, a transmembrane domain and a highly conserved but functionally uncharacterized cytoplasmic region. Like the other neural members of the immunoglobulin superfamily, Bravo carries the HNK-1 carbohydrate epitope, and specifically like L1 and Ng-CAM, Bravo is found predominantly in the form of a heterodimer, an intact chain cleaved in identical locations in all three molecules into two non-covalently associating parts. These data present an interesting view of retinotectal optic fiber outgrowth: As optic fibers grow in dense fascicles towards the optic nerve exit, they express both the known cell adhesion molecule Ng-CAM, and the closely related Bravo. As these fibers pass through the optic chiasm, Bravo is reduced on their surfaces, coincidentally in the same place and time that these fibers noticeably defasciculate, presumably to allow independent target seeking by individual axons. Furthermore, Bravo staining of retinal glial processes has been detected. The close relationhip of Bravo to Ng-CAM coupled to Ng-CAM's known homophilic binding capacity suggest Bravo may be the heterophilic glial ligand for Ng-CAM long postulated to exist. Bravo is identical in sequence with the recently characterized Ng-CAM related glycoprotein, Nr-CAM

Receptor-targeted co-transport of DNA and magnetic resonance contrast agents

Background: Ligand molecules conjugated to polylysine can be electrostatically bound to DNA and can bind receptors or antigens on the surface of cells, delivering the DNA into specific cells and tissues. Several researchers have used this approach to generate non-viral vehicles for the efficient delivery of DNA to specific cells. We have attempted to adopt this general approach to the cell-specific delivery of magnetic contrast agents for use in magnetic resonance imaging (MRI). Results: We have synthesized a new class of agents capable of both transfecting genes into cells and enhancing the contrast of the targeted cells for MRI. DNA is used both to encode a marker gene and as a molecular scaffold, which electrostatically binds polylysine conjugated to transferrin, an iron uptake protein, and polylysine modified with gadolinium chelated to diethylenetriaminepetaacetic acid. When cells displaying the transferrin receptor are treated with these particles, high levels of gene expression are observed, higher than with control particles composed only of transferrin, polylysine and DNA. The treated cells show specific MRI contrast enhancement, which did not require expression of the marker gene. Conclusions: The development of this class of particles permits the use of novel protocols by which genes for genetic therapy and agents for MRI contrast are cotransported. These protocols may allow non-invasive MRI monitoring of DNA delivery for gene therapy in real time