Biomedical subjects

R J Giger

Publications and source records attributed to R J Giger.

25 records · Page 2Linked to original sources

PubMed1996

Anatomy of rat semaphorin III/collapsin-1 mRNA expression and relationship to developing nerve tracts during neuroembryogenesis.

Semaphorin III/collapsin-1 (semaIII/coll-1) is a chemorepellent that exhibits a repulsive effect on growth cones of dorsal root ganglion neurons. To identify structures that express semaIII/coll-1 in developing mammals, we cloned the rat homologue and performed in situ hybridization on embryonic, neonatal, and adult rats. The relationship between semaIII/coll-1 mRNA distribution and developing nerve tracts was studied by combining in situ hybridization with immunohistochemistry for markers of growing nerve fibers. At embryonic day 11, semaIII/coll-1 expression was restricted to the olfactory pit, the basal and rostral surface of the telencephalic vesicle, the anlage of the eye, the epithelium of Rathke's pouch, and the somites. At later developmental stages, semaIII/coll-1 mRNA was found to be widely distributed in neuronal as well as in mesenchymal and epithelial structures outside the nervous system. Strong expression was found in the olfactory bulb, retina, lens, piriform cortex, amygdalostriatal area, pons, cerebellar anlage, motor nuclei of cranial nerves, and ventral spinal cord. After birth, mesenchymal staining decreased rapidly and expression became progressively restricted to specific sets of neurons in the central nervous system (CNS). In the mature CNS, semaIII/coll-1 mRNA remains detectable in mitral cells, neurons of the accessory bulb and cerebral cortex, cerebellar Purkinje cells, as well as a subset of cranial and spinal motoneurons. The temporal and spatial expression pattern of semaIII/coll-1 mRNA and its relationship to emerging nerve tracts suggests that semaIII/coll-1 is involved in guiding growing axons towards their targets by forming a molecular boundary that instructs axons to engage in the formation of specific nerve tracts.

Animals↗

PubMed1996

Continuous renewal of the axonal pathway sensor apparatus by insertion of new sensor molecules into the growth cone membrane.

BACKGROUND: Growth cones at the tips of growing axons move along predetermined pathways to establish synaptic connections between neurons and their distant targets. To establish their orientation, growth cones continuously sample for, and respond to, guidance information provided by cell surfaces and the extracellular matrix. To identify specific guidance cues, growth cones have sensor molecules on their surface, which are expressed differentially during the temporospatial progress of axon outgrowth, at levels that depend on the pattern of neural activity. However, it has not been elucidated whether a change in gene expression can indeed change the molecular composition and, hence, the function of the sensor apparatus of growth cones. RESULTS: We have constructed adenoviral gene transfer vectors of the chicken growth cone sensor molecules axonin-1 and Ng-CAM. Using these vectors, we initiated the expression of axonin-1 and Ng-CAM in rat dorsal root ganglia explants during ongoing neurite outgrowth. Using specific surface immunodetection at varying time points after infection, we found that axonin-1 and Ng-CAM are transported directly to the growth cone and inserted exclusively in the growth cone membrane and not in the axolemma of the axon shaft. Furthermore, we found that axonin-1 and Ng-CAM do not diffuse retrogradely, suggesting that the sensor molecules are integrated into multimolecular complexes in the growth cone. CONCLUSIONS: During axon outgrowth, the pathway sensor apparatus of the growth cone is continuously updated by newly synthesized sensor molecules that originate directly from the transcription/translation machinery. Changes in the expression of sensor molecules may have a direct impact, therefore, on the exploratory function of the growth cone.

Animals↗

PubMed1996

Implications for the domain arrangement of axonin-1 derived from the mapping of its NgCAM binding site.

The neuronal cell adhesion molecule axonin-1 is composed of six immunoglobulin and four fibronectin type III domains. Axonin-1 promotes neurite outgrowth, when presented as a substratum for neurons in vitro, via a neuronal receptor that has been identified as the neuron-glia cell adhesion molecule, NgCAM, based on the blocking effect of polyclonal antibodies directed to NgCAM. Here we report the identification of axonin-1 domains involved in NgCAM binding. NgCAM-conjugated microspheres were tested for binding to COS cells expressing domain deletion mutants of axonin-1. In addition, monoclonal antibodies directed to axonin-1 were assessed for their ability to block the axonin-1-NgCAM interaction, and their epitopes were mapped using the domain deletion mutants. The results suggest that the four amino-terminal immunoglobulin domains of axonin-1 form a domain conglomerate which is necessary and sufficient for NgCAM binding. Surprisingly, NgCAM binding to membrane-bound axonin-1 was increased strongly by deletion of the fifth or sixth immunoglobulin domains of axonin-1. Based on these results and on negative staining electron microscopy, we propose a horseshoe-shaped domain arrangement of axonin-1 that obscures the NgCAM binding site. Neurite outgrowth studies with truncated forms of axonin-1 show that axonin-1 is a neurite outgrowth-promoting substratum in the absence of the NgCAM binding site.

Animals↗

PubMed1995

The human TAX1 gene encoding the axon-associated cell adhesion molecule TAG-1/axonin-1: genomic structure and basic promoter.

The human TAX-1 gene (HGMW-approved symbol TAX1) is located on chromosome 1 (1q32.1) and encodes the neuronal cell adhesion molecule TAG-1/axonin-1. The gene product, termed TAG-1 in the rat and axonin-1 in the chicken, is composed of six immunoglobulin (Ig)-like and four fibronectin type III (FNIII)-like domains. It is found predominantly on the axons of particular nerve fiber tracts during neural development, and it has been demonstrated to function as a potent substratum for neurite outgrowth in vitro. Here we report the cloning and structural characterization of the TAX-1 gene. The transcribed region of the TAX-1 gene extends over about 40 kb. Like its chicken homologue, the human TAX-1 gene consists of 23 exons. Two GT/CA microsatellites were localized in the first intron; a polymorphism was found for one of them. Reporter gene analysis with serially truncated fragments of the 5'-flanking region indicated that a 164-bp fragment located immediately upstream of the putative transcription initiation site was sufficient to function as a basal promoter.

Animals↗

PubMed1995

The gene of chicken axonin-1. Complete structure and analysis of the promoter.

We have isolated and characterised the gene encoding the chicken axonal cell adhesion molecule axonin-1. This gene comprises 23 exons distributed over approximately 40 kb. Each of the six immunoglobulin-like domains and the four fibronectin-type-III-like domains of axonin-1 is encoded by two exons. The introns between two domains are exclusively phase I. Their exon/intron borders correspond to the domain borders of the protein, suggesting that the gene of axonin-1 had been generated by exon shuffling. Three transcripts with a length of 4.3 kb, 5 kb, and 8 kb are found, and we provide evidence that they result from alternative use of polyadenylation signals. In situ hybridization revealed co-localisation of these transcripts in time and space in the developing chicken retina. Several identical transcription initiation sites were found in retina, brain, and cerebellum by RNase protection assay and anchored polymerase chain reaction. By transfection of HeLa cells, rat PC-12 phaeochromocytoma cells, and chicken embryonic fibroblasts with serially truncated segments of the 5'-flanking region linked to a luciferase reporter gene, we have found that the sequence from -91 to +56 relative to the transcription initiation site is sufficient to promote efficient gene expression. Tissue-specific expression of the axonin-1 gene seems to be regulated in part by sequences more than 1 kb upstream of the transcription initiation site. As revealed by computer analysis, the sequence immediately upstream of exon 1 contains an AP-2 binding site, a tumor phorbol-ester-responsive element, and a homeodomain protein binding site, but no canonical TATA box. A second AP-2 binding site and a homeodomain protein binding site are located within exon 1.

Amino Acid Sequence↗

PubMed1995

Binding between the neural cell adhesion molecules axonin-1 and Nr-CAM/Bravo is involved in neuron-glia interaction.

Neural cell adhesion molecules of the immunoglobulin superfamily mediate cellular interactions via homophilic binding to identical molecules and heterophilic binding to other family members or structurally unrelated cell-surface glycoproteins. Here we report on an interaction between axonin-1 and Nr-CAM/Bravo. In search for novel ligands of axonin-1, fluorescent polystyrene microspheres conjugated with axonin-1 were found to bind to peripheral glial cells from dorsal root ganglia. By antibody blockage experiments an axonin-1 receptor on the glial cells was identified as Nr-CAM. The specificity of the interaction was confirmed with binding studies using purified axonin-1 and Nr-CAM. In cultures of dissociated dorsal root ganglia antibodies against axonin-1 and Nr-CAM perturbed the formation of contacts between neurites and peripheral glial cells. Together, these results implicate a binding between axonin-1 of the neuritic and Nr-CAM of the glial cell membrane in the early phase of axon ensheathment in the peripheral nervous system.

Animals↗

PubMed1992

A mutant of human insulin-like growth factor II (IGF II) with the processing sites of proinsulin. Expression and binding studies of processed IGF II.

A mutant of human insulin-like growth factor II (IGF II) was constructed by site-directed mutagenesis: the nucleotides coding for Ser33 and Ser39 were changed to yield Arg and Lys, respectively, thus creating two pairs of basic residues, Arg-Arg and Lys-Arg, as flanking sequences of the remaining C domain. [Arg33, Lys39]IGF II was expressed in NIH-3T3 cells as a processed two-chain peptide with a deletion of amino acid residues 37-40 and crosslinked by three disulfide bonds. This des(37-40)[Arg33]IGF II showed 3.6-fold and 7.4-fold reduced affinities to the type 1 and type 2 IGF receptor overexpressing cells, respectively, whereas the thymidine incorporation potency was the same as that of wild-type IGF II. We speculate that the discrepancy between the reduced binding to the type 1 IGF receptor and the full thymidine incorporation potency is due to the 6.1-fold reduced affinity of the expressed mutant to the co-expressed IGF binding protein 3 (IGFBP-3). The results suggest that des(37-40)[Arg33]IGF II assumes a conformation very similar to IGF II, and that the entire length of the C domain is not essential for biological activity.

3T3 Cells↗