Biomedical subjects

M M Portier

Publications and source records attributed to M M Portier.

At least 37 records · Page 2Linked to original sources

PubMed1992

Structure of the mouse gene encoding peripherin: a neuronal intermediate filament protein.

The gene encoding mouse peripherin, a neuronal intermediate filament protein, has been cloned. Its sequence, through 1021 nucleotides composing the 5'-flanking region, nine exons, eight introns and 547 nucleotides of the 3'-flanking region, as well as its transcription initiation site have been determined. The amino acid coding sequence differs from that of the rat peripherin gene. The mouse gene has an additional histidine near the N-terminal end, and shows three conservative and two non-conservative changes. The promoter sequence, containing the binding sites for transcription factors as well as other sequences is homologous to promoter regions of other type III intermediate filament protein genes and other neuronal-specific genes.

Amino Acid Sequence↗

PubMed1992

Autoantibodies against pancreatic beta-cells: characterization by western blot analysis in the non-obese diabetic (NOD) mouse.

The NOD mouse is a relevant model for studying autoimmune diabetes. As in human insulin-dependent diabetes mellitus, the nature of the autoantigen towards which the immune system is directed remains to be clarified. It has been shown that T cells are central to the disease process. However, autoantibodies may be used as a probe to identify islet autoantigens to which self tolerance is defective. Using Western blot analysis, we characterized autoantibodies which are specific for a 58 kDa islet antigen and a 29 kDa antigen. The 58 kDa autoantigen was present in cellular extracts prepared from rat tumoral insulin-secreting cells (Rin5F) and NOD islets but not from most other non-insulin-secreting cell lines. By contrast the 29 kDa antigen was a ubiquitous antigen expressed in all cell lines tested and was not further characterized since it is very likely to be responsible for secondary immunization rather than play any role in the NOD disease process. Anti-58 kDa autoantibodies were detected in all diabetic male and female NOD animals as well as in sera from old non-diabetic NOD animals. Anti-58 kDa antibodies were not detected in sera from young NOD mice (less than 6 weeks of age) or in sera from other conventional laboratory strains of mice including autoimmune prone animals such as MRL/lpr and (NZB x NZW)F1 mice. A monoclonal antibody (72.2) specific for the 58 kDa structure was obtained, which allowed further characterization of the corresponding islet cell antigen. The expression of the 58 kDa antigen was evidenced by Western blot analysis in normal islets and in a mouse neuroblastoma cell line.

Animals↗

PubMed1992

Chromosomal localisation of the mouse and human peripherin genes.

Using a mouse cDNA probe encoding for the major part of peripherin, a type III intermediate filament protein, we have assigned, by in situ hybridization, the mouse and human peripherin genes, Prph, to the E-F region of chromosome 15 and to the q12-q13 region of chromosome 12, respectively. These regions are known as homologous chromosomal segments containing other intermediate filament genes (keratins) and also other genes which could be co-ordinately regulated.

Animals↗

PubMed1992

[Neuronal cytoskeleton: structural, functional and dynamic aspects].

The cytoskeleton of practically every type of cell is constituted by 3 types of filaments: microfilaments, microtubules and intermediate filaments. The latter are cell-type specific and are represented in neurons by neurofilaments accompanied by other intermediate filaments, which have recently been described and whose nature depends on the period of development and on the neuronal type. Numerous studies demonstrate that many proteins are associated with microfilaments and microtubules and that they are responsible not only for the structure of the lattices but also for their function and dynamics. Moreover, these proteins can link one type of filament to another, and the network thus constituted, to the plasmic membrane and probably also to the nuclear membrane. The neuronal cytoskeleton is not static; it has, of course, a very important role in the maintenance of the structural integrity of the neuron, but it is also the promoter of axonal growth, the substratum against which many organelles move and thus has an essential role in axonal transport. In a few neuropathies chosen as examples for a disturbed axonal transport, abnormal bundles of filaments are observed; they are probably the end product of a primary disorder which has still to be analyzed.

Axons↗

PubMed1991

Network antibodies identify nuclear lamin B as a physiological attachment site for peripherin intermediate filaments.

We studied the molecular associations between peripherin (a neuronal, type III intermediate filament subunit) and nuclear lamins. We show here that isolated peripherin binds selectively to mammalian lamin B under in vitro conditions. We further demonstrate that a synthetic peptide, representing the proximal part of peripherin's tail domain (P1), also associates with mammalian lamin B in a saturable, cooperative, and specific fashion. Laboratory animals immunized with P1 spontaneously develop idiotypic and anti-idiotypic antibodies recognizing peripherin and lamin B, respectively. These data provide essentially in vivo evidence that lamin B represents a constitutive nuclear "receptor" site for the tail domains of peripherin intermediate filaments.

Amino Acid Sequence↗

PubMed1991

Glial fibrillary acidic protein immunoreactivity in adrenocortical and Leydig cells of the Syrian golden hamster (Mesocricetus auratus).

In an immunocytochemical investigation of the expression of glial fibrillary acidic protein (GFAP) in non-nervous system tissues ten anti-GFAP antibodies were used on a range of normal adult organs from different species. All four polyclonal and six monoclonal antibodies revealed the expression of GFAP in cells of the zona fasciculata and reticularis of the adrenal cortex and Leydig cells of the Syrian hamster. The Chinese hamster, mole, rat, mouse, guinea pig, rabbit, pig, duck and man were negative. Co-expression of immunoreactivity for GFAP and vimentin was observed in adrenocortical and Leydig cells of the Syrian hamster but there were differences in the staining patterns of these intermediate filament proteins. Expression of GFAP in adrenal cortex of Syrian hamster is confirmed by immunoblot and limited proteolysis analysis which reveal a light form which is immunochemically indistinguishable from its counterpart in the central nervous system. The results presented here suggest a new model for the study of the possible role of GFAP expression in cells known to be sites of steroid synthesis.

Adrenal Cortex↗

PubMed1991

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.

Adrenal Medulla↗

PubMed1990

Neuronal intermediate filaments in rat dorsal root ganglia: differential distribution of peripherin and neurofilament protein immunoreactivity and effect of capsaicin.

Two major neuronal populations were revealed in rat dorsal root ganglia, immunoreactive for either peripherin, or neurofilament triplet proteins (adult L2 ganglia: 66.2% and 25.6%, mainly small and large diameter cells, respectively), together with a minor, double-immunostained population (L2: 8.1%, mainly intermediate-size neurons). After capsaicin treatment, a striking expansion in the latter population was seen (L2: 22.0%) together with a significant increase in size, restricted to the same population and the (remaining) peripherin-only immunoreactive neurons. Calcitonin gene-related peptide (CGRP) immunoreactivity was revealed in neurons of all 3 groups, in both normal and capsaicin-treated rats.

Animals↗

PubMed1990

Cytokeratins as molecular markers in the evaluation of the precise differentiation stage of human gingival epithelium reconstituted in vitro.

Cytokeratins are considered to be molecular markers for different types of epithelial differentiation. They were used to investigate the precise differentiation stage of gingival epithelium, reconstituted in vitro, following two different culture procedures. Human trypsin-dissociated gingival keratinocytes were seeded either on a feeder layer of irradiated mouse 3T3 fibroblasts or on a connective tissue equivalent (lattice) made up of human fibroblasts in a collagen gel. The cytokeratins were extracted and analysed by two-dimensional gel electrophoresis. Although both methods showed on histological sections that cultured gingival keratinocytes formed a multilayered non-keratinizing epithelium, the cytokeratins patterns showed great differences. The gingival epithelium-like structure reconstituted on 3T3 feeder layer expressed some cytokeratins characteristic of the in situ gingival epithelium (K 5, 6, 14, 16, 17) and some which do not exist in the normal tissue (K 8, 18, 19, traces of K 13 and K 15) and are specific for embryonic, simple and tumour epithelia. However, the gingival epithelium reconstituted on connective tissue equivalent expressed all the cytokeratins present in the normal tissue (K 5, 6, 14, 16, 17), except those specific for terminal differentiation (K 1, 2, and 10/11). These findings suggest that the culture of gingival keratinocytes on connective tissue equivalents allows them to reproduce physiological stages of differentiation.

Biomarkers↗

PubMed1990

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.

Animals↗

PubMed1989

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.

Animals↗

PubMed1989

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.

Animals↗

PubMed1989

Multiple mRNAs encode peripherin, a neuronal intermediate filament protein.

Three cDNA clones of 1.6 (3u), 1.2 (5g) and 0.6 (5b) kbp, specific for peripherin, a neuronal intermediate filament protein (IFP), have been isolated from a murine neuroblastoma cell lambda gt11 library by immunoscreening using peripherin antiserum. Antibodies eluted from the fusion proteins produced by clones 3u and 5g recognize the peripherin spots on immunoblots. Where they overlap the three cDNAs have identical sequences. cDNA 5g exhibits the closest homology to type III IFP cDNAs. cDNA 3u is identical to the corresponding region of cDNA 5g, except for the insertion of a 96 bp fragment at a position corresponding to the junction of exons 4 and 5 in type III IFP cDNAs. cDNA 5b is also identical to the corresponding region of cDNA 5g, except for the deletion of a 62 bp fragment at the junction of exons 8 and 9 in type III IFP cDNAs. S1 mapping experiments performed with probes covering the 3' end of the two unexpected regions show that three distinct mRNAs correspond to the three cDNAs. Moreover, three peripherin products, two minor 61 and 56 kd products in addition to the major 58 kd peripherin, are observed when poly(A)+ RNA is in vitro translated, the 61 kd peripherin being translated from the 3u-selected RNA. The three RNAs originate from alternative splicing of a unique peripherin gene, thus generating polymorphism of peripherin.

Amino Acid Sequence↗

PubMed1989

Immunocytochemical localization of the intermediate filament protein peripherin in adult mouse adrenal chromaffin cells in culture.

Peripherin is the main intermediate filament protein in sympathetic neurons. Immunoreactivity to peripherin was studied in mouse adrenal chromaffin cells after 6 days in culture, and compared to immunoreactivity to tyrosine hydroxylase used as a general marker of chromaffin cells in culture. Most of the cells immunoreactive to tyrosine hydroxylase were rounded, with a glandular phenotype and a few of them had processes. The cells reactive to peripherin only constituted a small proportion of the chromaffin cells (2%), and most of them sent out processes. However, not all the cells with processes were reactive for peripherin. These results did not change in the presence of nerve growth factor. The discussion focuses on the significance of the sub-population of cells reactive to peripherin. We suggest that these cells resemble the small granule chromaffin cells, regarded as an intermediate cell type between glandular cells and neurons. The cells that expressed peripherin here are compared to those selected to form the PC12 clone. The presence of peripherin in only a few of the cells sending out neurite-like processes is discussed in relation to the expression of other neurofilament proteins in developing cells and to the influence of non-chromaffin cells.

Adrenal Medulla↗

PubMed1989

[Reconstitution in vitro of human gingiva].

A model of human gingiva to be used in pharmacological, basic and clinical research was performed in vitro. This model was obtained through a method of low density seeding epithelialization, from a seeding made up of dissociated human gingiva epithelial cells, of a connective tissue equivalent composed of human fibroblasts included in a collagen gel. The histological and ultrastructural data show a multilayered epithelium and the biochemical analysis (two dimensional gel electrophoresis known as NEPHGE) of the cytokeratins used as molecular markers for epithelial differentiation shows the precise differentiation state of the epithelium thus reconstituted. Even though this model has less of a differentiation than that of an in vivo gingival epithelium, it does actually reproduce exactly the structures of the human gingiva namely a multilayered epithelium lying on a connective tissue. It also offers the advantage of cellular elements which are compatible with gingival graftings.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1988

Characterization of two antigens in parkinsonian Lewy bodies.

Two antigens, G7 and G9, which are labelled by monoclonal antibodies in Lewy bodies in brains from patients with Parkinson's disease, were characterized by two-dimensional electrophoresis, followed by electroblotting, in order to explore their possible relationship with neuronal degeneration in this disease. The G7 antigen was found in the substantia nigra of subjects with Parkinson's disease and progressive supranuclear palsy, as well as in normal subjects. It was also found in the dopaminergic nucleus paranigralis and the locus coeruleus. The G9 antigen was found in the substantia nigra and locus coeruleus, but also in the caudate nucleus, terminal region of the nigrostriatal dopaminergic neurons, and in the cortex and cerebellum, terminal regions of the noradrenergic neurons in the locus coeruleus. The identity of the antigens remains unknown. They do not correspond to tyrosine hydroxylase or neurofilaments previously detected in Lewy bodies, or to other cytoskeletal proteins. Nor are they related to the presence of neuromelanin in the cells that degenerate in Parkinson's disease. The proteins, or at least the epitopes labelled by the antibodies, are found in normal brain, suggesting that these proteins do not play a causal role in the formation of the Lewy bodies in degenerating neurons in Parkinson's disease. The G7 antigen is absent from the cholinergic substantia innominata, where Lewy bodies are also found, indicating that these antigens are not essential for the formation of the corpuscle.

Antigens↗

PubMed1988

[Comparative expression of 2 intermediate filament proteins, peripherin and the 68 kDa neurofilament protein, during embryonal development of the 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.

Animals↗

PubMed1987

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.

Animals↗