Cytoskeletal protein synthesis and organization in cultured mouse osteoblastic cells. Effects of cell density.

The most abundant cytoskeletal proteins synthesized in mouse endosteal osteoblastic cells were identified employing two-dimensional polyacrylamide gel electrophoresis and immunoblotting. The relative rate of synthesis of the proteins were measured on radioautograms of detergent-soluble and -insoluble lysates of the cells labeled with [35S]methionine. Doubling initial cell density induced a 10-45% reduction in the de novo synthesis of actin, alpha-actinin, vimentin and beta-tubulins with no change in alpha-tubulins. Increasing cell density caused a 45% decrease in the polymerized form a actin with no change in the unpolymerized fraction, suggesting a correlation of alteration of the organization and synthesis of proteins.

A new monoclonal antibody recognizing the amino-terminal consensus sequence of vertebrate intermediate filament proteins.

The mouse monoclonal antibody ME 101 raised against human peripherin, an intermediate filament protein (IFP) specific to well defined neuronal populations, recognizes all the major classes of vertebrate IFP in immunoblotting assays. Desmin, GFAP, vimentin, peripherin and the lightest neurofilament protein (NF-L) were cleaved into carboxy- and amino-terminal halves by N-chlorosuccinimide at their unique trytophan residue. Whereas the antibody directed against the epitope common to every IFP (intermediate filament antigen or IFA) and located on the carboxy-terminal end of the rod domain recognizes the carboxy-terminal half, the ME 101 antibody, as the present study illustrates, recognizes specifically the amino-terminal half. From the amino acid sequence data of IFP, it is deduced that the cognate epitope is localized on the amino-terminal part of coil la.

Immunohistochemical evidence for the absence of central neuron projection to pial blood vessels and dura mater.

The present work shows that, in rat pial vessels and dura mater, all the nerve fibres observable by confocal fluorescence microscopy belong to the peripheral nervous system. It has been postulated that central nervous structures such as the raphe nuclei and the locus coeruleus could send direct projections to meningeal blood vessels. Mature neurons, whose perikaryons and axons are entirely located within the central nervous system, express the low molecular mass neurofilament protein and not the 57,000 mol. wt intermediate filament protein called "peripherin". This is the case for both raphe nuclei and locus coeruleus neurons [Leonard et al. (1988) J. Cell Biol. 106, 181-193]. Neurons which send axons outside the central nervous system or ganglionic neurons of the peripheral nervous system systematically express both proteins [Portier et al. (1984) Devl Neurosci. 6, 335-344]. Double labelling of pial vessels and meningeal tissue with antibodies directed against low molecular mass neurofilament and peripherin revealed nerve fibres immunoreactive to both antibodies and no nerve fibres reactive only to the low molecular mass neurofilament antibody. Conversely, cortical nerve fibres were immunoreactive only to the low molecular mass neurofilament antibody. It is thus concluded that the raphe nuclei and the locus coeruleus do not directly innervate meningeal tissues and, therefore, that these nuclei cannot directly intervene in cerebrovascular pathologies such as migraine headache or vasospasm. Secondarily, the present work also allowed for the first time the accurate observation of the spatial organization of the complete cerebrovascular innervation. Three main types of nerves can be defined on a morphological basis. A high proportion of these nerve fibres, either isolated or grouped in bundles, are varicose nerve fibres. Contacts between adjacent varicosities of the same type, which have been occasionally observed by electron microscopy, appear to be a very frequent feature.

Trophic action of pharmacological substances with a guanidine group on mouse neuroblastoma cells and chick ganglionic neurons in culture.

A series of substances (designated CTQ compounds) with a guanidine group have been synthesized and tested for their ability to promote neuronal survival and neurite outgrowth. Mouse neuroblastoma clonal cell lines grown in serum-containing medium for 10 days as well as primary cultures of embryonic chicken ganglion neurons grown in serum-free defined medium for 1 or 2 days have been used for the experiments. Among the various CTQ compounds (CTQ1-CTQ20) tested, only CTQ8 exerted positive neurotrophic effects on these peripheral neuronal cells. At a concentration of 10(-4) M, CTQ8 enhanced neuritogenesis of neuroblastoma cells. However, the most striking influence of CTQ8 was its promoting effect (6- to 10-fold) on the survival of chicken ciliary and dorsal root ganglionic neurons at concentrations ranging from 10(-3) M to 5 x 10(-4) M.

Peripherin and neurofilaments: expression and role during neural development.

This review focuses attention on the expression of peripherin and the low-molecular mass neurofilament protein during development, as well as on recent results concerning the roles of these neuronal proteins. Peripherin is the only type III intermediate filament that has been shown to be expressed in neurons but exclusively in motor, sensory and sympathetic neurons; moreover, it is co-expressed with neurofilament proteins (NFP). Clearly, peripherin is expressed concomitantly with axonal growth during development, and its synthesis appears necessary to axonal regeneration in the adult. As to NFP, they are presumed to maintain the axonal diameter and thereby ensure a normal conduction velocity. In many neuropathies, either occurring in man or provoked by different means in animals, the neurofilament network is disrupted thus giving rise to bundles of filaments in perikarya or along axons; consequently, the axonal transport is impaired. The possible significance of the overexpression of NFP is discussed.

[Comparative expression of 2 intermediate filament proteins, peripherin and the 68 kDa neurofilament protein, during embryonal development of the rat]. / Expression comparée de deux protéines de filaments intermédiaires, la périphérine et la protéine de neurofilament 68 kDa, au cours du développement embryonnaire du rat.

Peripherin, an intermediate filament protein, was originally detected by biochemical methods in the neurons of the peripheral nervous system. We now studied its expression and cellular localization by immunocytochemical methods in the developing rat embryo, and compared them with the expression and localization of the 68 kDa neurofilament protein. It appears that peripherin is expressed not only in the neurons of the peripheral nervous system, but also in some well defined neuronal populations of the central nervous system. These results focus on the questions of the phylogenetic origin and of the function of peripherin.

Differential expression of two neuronal intermediate-filament proteins, peripherin and the low-molecular-mass neurofilament protein (NF-L), during the development of the rat.

The expression of peripherin, an intermediate filament protein, had been shown by biochemical methods to be localized in the neurons of the PNS. Using immunohistochemical methods, we analyzed this expression more extensively during the development of the rat and compared it with that of the low-molecular-mass neurofilament protein (NF-L), which is expressed in every neuron of the CNS and PNS. The immunoreactivity of NF-L is first apparent at the 25-somite stage (about 11 d) in the ventral horn of the spinal medulla and in the posterior part of the rhombencephalon. The immunoreactivity of peripherin appears subsequently, first colocalized with that of NF-L. Both immunoreactivities then spread out along rostral and caudal directions, but whereas the immunoreactivity of NF-L finally becomes noticeable in every part of the nervous system, that of peripherin remains localized to (1) the motoneurons of the ventral horn of the spinal medulla; (2) the autonomic ganglionic and preganglionic neurons; and (3) the sensory neurons. These results demonstrate that, in the neurons that originate from migrating neural crest cells, the immunoreactivities of peripherin and of NF-L become apparent only when they have reached their destination. The results also show that peripherin is expressed more widely than has been previously observed and that this protein occurs in neuronal populations from different lineages (neural tube, neural crest, placodes) with different functions (motoneurons, sensory and autonomic neurons). The common point of these neurons is that they all have axons lying, at least partly, at the outside of the axis constituted by the encephalon and the spinal medulla; this suggests that peripherin might play a role in the recognition of the axonal pathway through the intermediary of membrane proteins.

Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons.

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.

Origin of the beta cells of the islets of Langerhans is further questioned by the expression of neuronal intermediate filament proteins, peripherin and NF-L, in the rat insulinoma RIN5F cell line.

Intermediate filament proteins of the rat insulinoma RIN5F cell line were characterized. Two-dimensional gel analysis followed by immunostaining of proteins demonstrated that these cells express both peripherin and the low-molecular-mass neurofilament protein (NF-L); this was confirmed for peripherin by immunohistochemistry, peptide analysis and Northern blot. No expression of these proteins could be detected with these same methods either in the adult pancreas or in the tumor at the origin of the cell line, although such expression was apparent on sections of rat pancreas at embryonal day 16. These results were compared to those obtained on the rat pheochromocytoma PC12 cell line: expression in the adrenal medulla of the embryo, no expression either in the adult tissue or in the tumor, but solely in the derived cell line. The expression of neuronal intermediate filament proteins in the rat insulinoma RIN5F cell line is discussed in relation to its similarity in the rat pheochromocytoma PC12 cell line, and its meaning as to the developmental cell lineage; an ectodermal origin is suggested for the pancreatic islet cells.

The Na-G ion channel is transcribed from a single promoter controlled by distinct neuron- and Schwann cell-specific DNA elements.

Na-G is a putative sodium (or cationic) channel expressed in neurons and glia of the PNS, in restricted neuronal subpopulations of the brain, and in several tissues outside the nervous system, like lung and adrenal medulla. To analyze the mechanisms underlying tissue-specific expression of this channel, we isolated the 5' region of the corresponding gene and show that Na-G mRNA transcription proceeds from a single promoter with multiple initiation sites. By transgenic mice studies, we demonstrate that 600 bp containing the Na-G proximal promoter region and the first exon are sufficient to drive the expression of a beta-galactosidase reporter gene in neurons of both CNS and PNS, whereas expression in Schwann cells depends on more remote DNA elements lying in the region between -6,500 and -1,050 bp upstream of the main transcription initiation sites. Crucial elements for lung-specific expression seem to be located in the region between -1,050 and -375 bp upstream of the promoter. Using in vivo footprint experiments, we demonstrate that several sites of the Na-G proximal promoter region are bound specifically by nuclear proteins in dorsal root ganglion neurons, as compared with nonexpressing hepatoma cells.