29 research outputs found
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
Topologically Restricted Appearance in the Developing Chick Retinotectal System of Bravo, a Neural Surface Protein: Experimental Modulation by Environmental Cues
A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. Axons growing out from retinal explants on retinal basal lamina, their natural substrate, express Bravo, whereas such axons growing on collagen do not. Retinal explants provide a valuable system to characterize the mechanism of Bravo restriction, as well as the cellular signals controlling it. Bravo was identified with monoclonal antibodies from a collection generated against exposed molecules isolated by using a selective cell surface biotinylation procedure. The NH2-terminal sequence of Bravo shows similarity with L1, a neural surface molecule which is a member of the immunoglobulin superfamily. This possible relationship to L1, together with its restricted appearance, suggests an involvement of Bravo in axonal growth and guidance
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
Uridine-Conjugated Ferrocene DNA Oligonucleotides:Â Unexpected Cyclization Reaction of the Uridine Base
NrCAM, a neuronal system cell-adhesion molecule, is induced in papillary thyroid carcinomas
NrCAM (neuron-glia-related cell-adhesion molecule) is primarily, although not solely, expressed in the nervous system. In the present study, NrCAM expression was analysed in a series (46) of papillary thyroid carcinomas (PTCs) and paired normal tissues (NT). Quantitative reverse transcriptase (QRT)-PCR revealed that NrCAM expression was upregulated in all PTCs compared to normal thyroid, whatever the stage or size of the primary tumour. NrCAM transcript levels were 1.3- to 30.7-fold higher in PTCs than in NT. Immunohistochemistry (IHC) confirmed that the expression of NrCAM was considerably higher in tumours (score 2+/3+) than in adjacent normal paratumoural thyroid tissue. The NrCAM protein was detected in all but three (93.3%) PTC samples, and it was mainly cytoplasmic; in some cases there was additional membranous localisation – basolateral and partly apical. In the normal thyroid and tissues surrounding tumours, focal NrCAM immunolabelling was seen only in follicles containing tall cells, where staining was restricted to the apical pole of thyrocytes. Western blot analysis corroborated the QRT–PCR and IHC results, showing higher NrCAM protein levels in PTCs than in paired NT. The level of overexpression of the NrCAM mRNA in tumourous tissue appeared to be independent of the primary tumour stage (pT) or the size of the PTC. These data provide the first evidence that NrCAM is overexpressed in human PTCs at the mRNA and protein levels, whatever the tumour stage. Thus, the induction and upregulation of NrCAM expression could be implicated in the pathogenesis and behaviour of papillary thyroid cancers
Neogenin, an avian cell surface protein expressed during terminal neuronal differentiation, is closely related to the human tumor suppressor molecule deleted in colorectal cancer.
Transfection of Folate-Polylysine DNA Complexes: Evidence for Lysosomal Delivery
We are utilizing the folate receptor for the intracellular delivery of DNA. In this study, a folate-poly-L-lysine (FPLL) conjugate was synthesized and equilibrated with plasmid DNA encoding the firefly luciferase gene. The FPLL-DN_A complexes were added to KB cells treated with chloroquine. Luciferase activity of cells incubated with FPLL-DNA was 6-fold higher than of cells exposed to poly-L-lysine. (PLL)-DNA. The addition of free folic acid competitively inhibited the enhancement of gene
expression. Removal of chloroquine from the media significantly inhibited transfection efficiency of
FPLL-DNA complexes. We conclude that FPLL-DNA complexes are delivered into KB cells via folate receptor-mediated endocytosis and likely follow a lysosomal pathway into the cytoplasm
Transfection of Folate-Polylysine DNA Complexes: Evidence for Lysosomal Delivery
We are utilizing the folate receptor for the intracellular delivery of DNA. In this study, a folate-poly-L-lysine (FPLL) conjugate was synthesized and equilibrated with plasmid DNA encoding the firefly luciferase gene. The FPLL-DN_A complexes were added to KB cells treated with chloroquine. Luciferase activity of cells incubated with FPLL-DNA was 6-fold higher than of cells exposed to poly-L-lysine. (PLL)-DNA. The addition of free folic acid competitively inhibited the enhancement of gene
expression. Removal of chloroquine from the media significantly inhibited transfection efficiency of
FPLL-DNA complexes. We conclude that FPLL-DNA complexes are delivered into KB cells via folate receptor-mediated endocytosis and likely follow a lysosomal pathway into the cytoplasm