Neurofascin: a novel chick cell-surface glycoprotein involved in neurite-neurite interactions

We have identified neurofascin, a novel chick cell-surface glycoprotein involved in neurite-neurite interactions. Neurofascin is defined by its reactivity with monoclonal antibody (MAb) F6, which detects two polypeptides (160 and 185 kd) in immunotransfers of brain plasma membrane proteins. Immunoaffinity chromatography using immobilized MAb F6 yields major molecular mass bands at 185, 160, 135-110, and 92 kd. Fingerprint analyses show that these polypeptides are related. Neurofascin is expressed primarily in fiber-rich areas of embryonic cerebellum, spinal cord, and retina. Fab fragments of polyclonal antibodies to neurofascin interfere with the outgrowth of retinal and sympathetic axons in two different in vitro bioassays. Neurofascin is immunologically distinct from other known neurite-associated surface glycoproteins.     

Membrane glycoproteins involved in neurite fasciculation

Lectin affinity chromatography combined with mAb production was used to identify chick neural cell surface molecules related to L1 antigen, a mouse neural glycoprotein implicated in cell-cell adhesion (Rathjen, F. G., and M. Schachner, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1-10). A glycoprotein, G4 antigen, isolated by mAb G4 from adult chick brain is described which comprises a major 135-kD component, a minor doublet at 190 kD, and diffusely migrating bands at 80 and 65 kD in SDS PAGE. This molecule is structurally related to mouse L1 antigen according to NH2-terminal amino acid sequence (50% identity) as well as the behavior of its components in two-dimensional IEF/SDS PAGE gels. A second chicken glycoprotein, F11 antigen, was isolated from adult chick brain using mAb F11. This protein has also a major 135-kD component and minor components at 170 kD and 120 kD. Both immunotransfer analysis with polyclonal antibodies to mAb G4 and to mAb F11 isolate and the behavior on IEF/SDS PAGE gels indicates that the major 135-kD component of F11 antigen is distinct from G4 antigen components. However, the 135-kD component of F11 antigen shares with G4 antigen and the neural cell adhesion molecule (NCAM) the HNK-1/L2 carbohydrate epitope. In immunofluorescence studies, G4 and F11 antigenic sites were found to be associated mainly with the surface of process-bearing cells, particularly in fiber-rich regions of embryonic brain. Although Fab fragments of polyclonal antibodies to mAbs G4 or F11 immunoaffinity isolate only weakly inhibit the Ca2+-independent aggregation of neural cells, they strongly inhibit fasciculation of retinal axons. Together these studies extend the evidence that bundling of axons reflects the combined effects of a group of distinct cell surface glycoproteins.     

Molecular diagnosis of Duchenne and Becker muscular dystrophies. Current data

Carrier diagnosis and prenatal diagnosis of Duchenne's muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD) has become possible using some twenty RFLPs detected by more than a dozen Xp21 probes that are either intragenic or flanking the disease locus. Results from familial studies on 88 DMD and BM families stress important considerations concerning a priori and final risks, individuals necessary for the identification of the phase, and the different strategies that can be applied, regardless of whether the study concerns an on-going pregnancy or a carrier-status determination, and whether the patient is at high or low risk. Finally, multiple sources of difficulties in interpreting the results depend on a) the occurrence of new mutations that must be traced; b) the existence of meiotic recombination; c) the necessity, in some instances, of relying upon the sole identification of the paternal X. These considerations emphasize the characteristics and the important limitations of this type of methodology.     

Biochemical characterization of polypeptide components involved in neurite fasciculation and elongation

Polypeptide components and carbohydrate linkage types of F11 antigen and G4 antigen, two chick cell-surface glycoproteins implicated in neurite fasciculation and elongation [Rathjen, F.G., Wolff, J.M., Bonhoeffer, F. and Rutishauser, U. (1987) J. Cell Biol. 104, 343-353], have been studied in comparison to mouse L1 antigen. Tryptic fingerprint analysis does not reveal any relation of the 130-kDa components of G4 or F11 antigens to each other or to neural cell-adhesion molecules. The 180/190-kDa component of G4 antigen comprises parts of the 130-kDa and 80/65-kDa components and shares a sequence corresponding to the amino terminus of the G4 130-kDa component as shown serologically with anti-peptide sera. This closely parallels the relationship found for mouse L1 antigen components. In contrast, the F11 170-kDa component is different from the F11 130-kDa component, as shown serologically and by fingerprint analysis. A combination of chemical and enzymatic deglycosylation methods reveals that while O-glycosylation cannot be detected F11 130-kDa, G4 130-kDa and L1 140-kDa components contain N-linked carbohydrates. Endoglycosidase H treatment shows that the oligosaccharides present in the G4 130-kDa component and mouse L1 are mostly of the complex type, while the F11 130-kDa component consists of two populations, one containing mainly complex-type carbohydrates and a second containing high-mannose/hybrid-type carbohydrates.     

Characterization of a human ''midisatellite'' sequence.

We have examined the structure and DNA sequence of a human genomic locus that consists of a large hypervariable region made up of repeats of a simple sequence. With several restriction enzymes, the locus shows many restriction fragments that vary quantitatively as well as qualitatively. Other restriction enzymes produce only a single, high-molecular-weight fragment at this locus. Almost all of the fragments are revealed with a simple sequence probe. Southern transfers of the high-molecular-weight restriction fragments produced by the restriction enzymes NotI and SfiI, resolved by pulsed-field gel electrophoresis, gave at most two fragments, demonstrated to be allelic, showing that the majority of the restriction fragments seen in the complex patterns are at a single locus. The estimated size of the region homologous to the probe varied from 250 to 500 kilobases. DNA sequencing indicated that the region consists of tandem repeats of a 40-base-pair sequence. Some homology was detected to the tandem repeating units of the insulin gene and the zetaglobin pseudogene hypervariable regions, and to the "minisatellite" DNA at the myoglobin locus.     

Selective illumination of single photoreceptors in the house fly retina: local membrane turnover and uptake of extracellular horseradish peroxidase (HRP) and Lucifer Yellow

Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.     

Membrane-damaging action of ricin on DPPC and DPPC-cerebrosides assemblies

Perturbations induced by a toxic lectin (ricin) on lipid organisation of model membranes prepared with DPPC and DPPC-cerebrosides mixtures have been analysed by Raman and infrared spectroscopy, two powerful and non-invasive methods. Our approach involves the observation of changes in the vibrational spectra of liquid multilayers in the PO ₂ ^- , C=0 and CH₂ spectral regions for two lipid: ricin molar ratios (225:1, 75:1).The interfacial and polar regions of the multilayers, analysed by FTIR, appear to be perturbed by the protein. With both kinds of membranes, ricin mainly perturbs the C=0 ester groups of the sn-2 acylchain of DPPC. In the PO ₂ ^- stretching region, the frequency shifts are correlated with changes in polar group hydration.In the hydrophobic core of the multilayer membrane studied by Raman spectroscopy, the interaction of ricin is associated with changes in lipid packing. These perturbations depend upon the lipid composition of the membrane. With DPPC membranes, an affect is detected at temperatures lower than T _m .It corresponds to a decrease of the lipid ordering. With DPPC-cer membranes, the protein increases the acylchain packing order regardless of the temperature of the experiments (10°C<T<75°C). No perturbation of T _m is observed after addition of ricin to either DPPC or DPPC-cer membranes.The different perturbations detected by Raman and FTIR suggest that ricin mainly interacts with the interfacial domains of the membranes.     

Changes in the cellular permeability of the embryonic axis inGcer arietinum L. seeds during germination

The effect of short chain fatty acids on the cellular permeability of embryonic axis inGcer arietinum seeds was studied. Octanoic (OCT) and nonanoic (NON) adds, which reduce both germination and growth of the embryonic axis and raise the inhibitor effects of the supraoptimal temperatures (30?C), induce a greater ionic efflux into the medium (conductivity). NON reduces glucose (3-0-MG) and K⁺ (⁸⁶Rb) uptake during the germinative process, this action being counteracted by fusicoccin (FC) at optimal (25?C) and supraoptimal temperatures (30 ?C). Tonoplast and plasmalemma increase their permeability to the K⁺ efflux when NON is present. Addition of NON+FC gives rise to higher values in the efflux rate, the vacuolar compartment being the most affected. Temperatures around zero (2 ?C) notably reduce the isotope efflux from cytosol and vacuole. NON acid does not significantly affect the efflux of³H-ABA, suggesting that it does not cause any important changes in the phytohormone compartmentation.