Drug exposure and the risk of multiple sclerosis: A systematic review.

BACKGROUND: Several environmental and lifestyle factors have been associated with multiple sclerosis (MS) risk, including some pharmacological treatments. We systematically reviewed the literature on prescription drug exposure and MS risk. METHODS: Six databases were searched for original observational studies reporting drug exposure and MS risk published before 2017. RESULTS: Thirteen articles fulfilled inclusion criteria. Exposure to neither amiloride nor valproic acid was associated with MS (adjusted hazard ratio (adj.HR = 1.34;95% CI:0.81-2.20; adj.HR = 1.30;95%CI:0.44-3.80, respectively). Four studies explored oral contraceptive exposure and reported no association with MS; while a single study found an increased risk (odds ratio [adj.OR] = 1.52;95%CI:1.21-1.91). While penicillin exposure was associated with reduced risk of developing MS (adj.OR = 0.5;95%CI:0.3-0.9), a later study observed an elevated risk for penicillin (adj.OR = 1.21;95%CI:1.10-1.27) and all antibiotics (adj.OR = 1.41;95%CI:1.29-1.53), which was potentially attributed to underlying infection. Anti-tumor necrosis factor-alpha (TNFα) was not associated with MS risk in persons with inflammatory bowel disease (standard morbidity ratio = 4.2;95%CI:0.1-23.0) and arthritis (standardized incidence ratio = 1.38;95%CI:0.69-2.77); however, men exposed to anti-TNFα who also had arthritis and individuals with ankylosing spondylitis were at an increased risk (standardized incidence ratios = 3.91;95%CI:1.47-10.42 and 3.48;95%CI:1.45-8.37, respectively). A reduced risk of MS was observed with exposure to the beta2-adrenergic agonist fenoterol (adj.OR = 0.58;95%CI:0.45-0.76), and the sedating histamine 1-receptor antagonists (adj.OR = 0.2;95%CI:0.1-0.8), but not the non-sedating equivalent (adj.OR = 0.8;95%CI:0.4-1.6). CONCLUSIONS: The suggestion that some drugs may prevent MS is intriguing and warrants further study. In addition, further pharmacovigilance is needed to assess the safety of anti-TNFα drugs in specific populations in the context of MS risk.

Cell-substratum adhesion in embryonic chick central nervous system is mediated by a 170,000-mol-wt neural-specific polypeptide.

Embryonal chick neural retina cells release into the culture medium a complex of proteins and glycosaminoglycans, termed adherons, that promote cell to substratum adhesion. A monoclonal antibody (C1H3) blocks adheron-mediated cell to substratum adhesion and specifically binds to a 170,000-mol-wt protein present in retinal adherons (Cole, G.J., and L. Glaser, 1984, J. Biol. Chem., 259:4031-4034). The 170,000-mol-wt protein also can be identified in embryonic chick brain and peripheral nervous tissue. In the neural retina, C1H3 also binds to a second antigen with a molecular weight of 140,000 that is absent in the brain. Embryonic brain, therefore, provides a source for the immunopurification of the 170,000-mol-wt protein. Brain adherons also contain the 170,000-mol-wt protein, and cell to substratum adhesion mediated by these adherons is blocked by the C1H3 monoclonal antibody. The 170,000-mol-wt protein in the brain is therefore functionally identical to that in the retina. To demonstrate that adheron-mediated cell to substratum adhesion is caused by cell binding to the 170,000-mol-wt protein, we showed that (a) protease digestion, but not glycosaminoglycan hydrolase digestion of adherons, blocked their ability to bind cells to substratum; (b) the immunopurified 170,000-mol-wt protein blocks adheron-mediated cell to substratum adhesion; and (c) cells can bind to immunopurified 170,000-mol-wt protein bound to glass surfaces.

A heparin-binding domain from N-CAM is involved in neural cell-substratum adhesion.

Cell-substratum adhesion in the embryonic chicken nervous system has been shown to be mediated in part by a 170,000-mol-wt polypeptide that is a component of adherons. Attachment of retinal cells to the 170,000-mol-wt protein is inhibited by the C1H3 monoclonal antibody and by heparan sulfate (Cole, G. J., D. Schubert, and L. Glaser, 1985, J. Cell Biol., 100:1192-1199). In the present study we have demonstrated that the 170,000-mol-wt C1H3 polypeptide is immunologically identical to the neural cell adhesion molecule N-CAM, and that the 170,000-mol-wt component of N-CAM is preferentially secreted by cells as a component of adherons. We have identified a monoclonal antibody, designated B1A3, that inhibits heparin binding to N-CAM and cell-to-substratum adhesion. A 25,000-mol-wt heparin (heparan sulfate)-binding domain of N-CAM has been identified by limited proteolysis, and this fragment promotes cell attachment when bound to glass surfaces. The fragment also partially inhibits cell binding to adherons when bound to retinal cells, and the B1A3 monoclonal antibody inhibits retinal cell attachment to substrata composed of intact N-CAM or the heparin-binding domain. These data are the first evidence that N-CAM is a multifunctional protein that contains both cell-and heparin (heparan sulfate)-binding domains.

Cell-substratum adhesion in chick neural retina depends upon protein-heparan sulfate interactions.

Embryonic chick neural retina cells in culture release complexes of proteins and glycosaminoglycans, termed adherons, which stimulate cell-substratum adhesion when adsorbed to nonadhesive surfaces. Two distinct retinal cell surface macromolecules, a 170,000-mol-wt glycoprotein and a heparan sulfate proteoglycan; are components of adherons that can independently promote adhesion when coated on inert surfaces. The 170,000-mol-wt polypeptide contains a heparin-binding domain, as indicated by its retention on heparin-agarose columns and its ability to bind [3H]heparin in solution. The attachment of embryonic chick retinal cells to the 170,000-mol-wt protein also depends upon interactions between the protein and the heparan sulfate proteoglycan, since heparan sulfate in solution disrupts adhesion of chick neural retina cells to glass surfaces coated with the 170,000-mol-wt protein. This adhesion is not impaired by chondroitin sulfate or hyaluronic acid, which indicates that inhibition by heparan sulfate is specific. Polyclonal antisera directed against the cell surface heparan sulfate proteoglycan also inhibit attachment of retinal cells to the 170,000-mol-wt protein, which suggests that cell-adheron binding is mediated in part by interactions between cell surface heparan sulfate proteoglycan and 170,000-mol-wt protein contained in the adheron particles. Previous studies have indicated that this type of cell-substratum adhesion is tissue-specific since retina cells do not attach to muscle adherons. Schubert D., M. LaCorbiere, F. G. Klier, and C. Birdwell, 1983, J. Cell Biol. 96:990-998.

Topographic localization of the heparin-binding domain of the neural cell adhesion molecule N-CAM.

Previous studies have reported that the cell-binding region of the neural cell adhesion molecule (N-CAM) resides in a 65,000-D amino-terminal fragment designated Frl (Cunningham, B. A., S. Hoffman, U. Rutishauser, J. J. Hemperly, and G. M. Edelman, 1983, Proc. Natl. Acad. Sci. USA, 80:3116-3120). We have reported the presence of two functional domains in N-CAM, each identified by a specific mAb, that are required for cell-cell or cell-substratum adhesion (Cole, G. J., and L. Glaser, 1986, J. Cell Biol., 102:403-412). One of these domains is a heparin (heparan sulfate)-binding domain. In the present study we have determined the topographic localization of the heparin-binding fragment from N-CAM, which has been identified by our laboratory. The B1A3 mAb recognizes a 25,000-D heparin-binding fragment derived from chicken N-CAM, and also binds to a 65,000-D fragment, presumably Frl, produced by digestion of N-CAM with Staphylococcus aureus V8 protease. Amino-terminal sequence analysis of the isolated 25,000-D heparin-binding domain of N-CAM yielded the sequence: Leu-Gln-Val-Asp-Ile-Val-Pro-Ser-Gln-Gly. This sequence is identical to the previously reported amino-terminal sequence for murine and bovine N-CAM. Thus, the 25,000-D polypeptide fragment is the amino-terminal region of the N-CAM molecule. We have also shown that the B1A3 mAb recognizes not only chicken N-CAM but also rat and mouse N-CAM, indicating that the heparin-binding domain of N-CAM is evolutionarily conserved among different N-CAM forms. Additional peptide-mapping studies indicate that the second cell-binding site of N-CAM is located in a polypeptide region at least 65,000 D from the amino-terminal region. We conclude that the adhesion domains on N-CAM identified by these antibodies are physically distinct, and that the previously identified cell-binding domain on Frl is the heparin-binding domain.

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides.

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.

Long-term behavioral impairment following acute embryonic ethanol exposure in zebrafish.

BACKGROUND: Developmental exposure to ethanol has long been known to cause persisting neurobehavioral impairment. However, the neural and behavioral mechanisms underlying these deficits and the importance of exposure timing are not well-characterized. Given the importance of timing and sequence in neurodevelopment it would be expected that alcohol intoxication at different developmental periods would result in distinct neurobehavioral consequences. METHODS: Zebrafish embryos were exposed to ethanol (0%, 1%, 3%) at either 8-10 or 24-27 h post-fertilization (hpf) then reared to adolescence and evaluated on several behavioral endpoints. Habituation to a repeated environmental stimulus and overall sensorimotor function were assessed using a tap startle test; measurements of anxiety and exploration behavior were made following introduction to a novel tank; and spatial discrimination learning was assessed using aversive control in a three-chambered apparatus. Overt signs of dysmorphogenesis were also scored (i.e. craniofacial malformations, including eye diameter and midbrain-hindbrain boundary morphology). RESULTS: Ethanol treated fish were more active both at baseline and following a tap stimulus compared to the control fish and were hyperactive when placed in a novel tank. These effects were more prominent following exposure at 24-27 hpf than with the earlier exposure window, for both dose groups. Increases in physical malformation were only present in the 3% ethanol group; all malformed fish were excluded from behavioral testing. DISCUSSION: These results suggest specific domains of behavior are affected following ethanol exposure, with some but not all of the tests revealing significant impairment. The behavioral phenotypes following distinct exposure windows described here can be used to help link cellular and molecular mechanisms of developmental ethanol exposure to functional neurobehavioral effects.

Localization of transitin mRNA, a nestin-like intermediate filament family member, in chicken radial glia processes.

We have examined the gene expression of two radial glia intermediate filament proteins, transitin and vimentin, in the developing chick CNS. Despite global similarities in their mRNA distributions, marked regional differences are observed. Most notably, we show that transitin mRNA is localized along radial glial processes and is localized to radial glia endfeet, whereas vimentin mRNA is not localized in radial glia. Localization of transitin mRNA is best shown in the diencephalic radial glia, as well as cerebellar Bergmann glia. In addition, in the early embryonic optic tectum, telencephalon, and retina, transitin mRNA is highly localized to radial glia endfeet, which is suggestive of its transport in these cells. These in vivo demonstrations of transitin mRNA localization are confirmed by in situ hybridization analysis of cultured chick brain radial glia, which demonstrates the presence of granular staining for transitin mRNA in glial processes. Transitin mRNA distribution in developing muscle also shows a highly regulated expression pattern, especially along the Z-lines of myofibrils. As further support for the transport and localization of transitin mRNA in radial glia and muscle, we have identified a consensus RNA transport signal in transitin mRNA that is absent from vimentin. These data suggest that the local regulation of transitin protein synthesis may contribute to its function as an intermediate filament protein in radial glia.

Distribution and substrate properties of agrin, a heparan sulfate proteoglycan of developing axonal pathways.

The distribution and substrate properties of agrin, an extracellular matrix heparan sulfate proteoglycan (HSPG), was investigated in the developing chick nervous system by immunocytochemistry, Western blotting, and in neurite outgrowth assays. By comparing the distribution of agrin with that of laminin-1, merosin (laminin-2), neurofilament, and neural cell adhesion molecule (NCAM), it was found that throughout development, agrin is a constituent of all basal laminae. From embryonic day (E) 4 onwards, agrin is also abundant in axonal pathways of the central nervous system, such as the optic nerve, the tectobulbar pathway, the white matter of the spinal cord, and the marginal and the molecular layers of the forebrain and the cerebellum. The abundance of agrin in brain decreases from E13 onwards. In the peripheral nervous system, agrin is present throughout development as a constituent of the Schwann cell basal laminae. Western blots confirmed the immunocytochemical data, showing maximum expression of agrin occurs during the early to medium stages of brain development. Western blots also showed that in mouse and human brain, agrin exists as an HSPG. Purified agrin did not support neurite outgrowth, rather it inhibited retinal neurite extension on mixed agrin/merosin substrates. Despite the fact that agrin, when used as a substrate inhibited neurite outgrowth, its temporal and spatial overlap with growing axons suggests that agrin has a supportive role in the development of axonal pathways, possibly as a binding component for growth factors and cell adhesion proteins.

Characterization of the chicken transitin gene reveals a strong relationship to the nestin intermediate filament class.

Our laboratory previously reported that transitin is a radial glial intermediate filament protein sharing the basic structural features common to all intermediate filament (IF) proteins. It contains an alpha-helical core domain flanked by a short nonhelical head and a long COOH-terminal tail. The core sequence of transitin shows the greatest similarity to Xenopus tanabin and to rat and human nestin. We also reported that transitin has multiple splice variants derived from the deletion or inclusion of a leucine-zipper heptad repeat domain in the COOH-terminal tail. In the present study, we provide new evidence to support the classification of nestin and transitin in the same group of IF proteins based on the number and position of its introns. In addition, we suggest that the different isoforms of transitin are produced by a splicing mechanism that recognizes consensus 5' and 3' splice sites contained within the coding sequence of the leucine-zipper heptad repeat domain.

An alternatively spliced, 5'-truncated MAP1B isoform is expressed in the developing chick nervous system.

Our laboratory has previously characterized a keratan sulfate proteoglycan, named claustrin, and shown by molecular cloning that claustrin and the mouse MAP1B protein share high homology, with claustrin representing a 5'-truncated fragment of MAP1B. In the present study, we examine further the relationship between claustrin and MAP1B, and also describe the isolation of a cDNA encoding the 3'-region of MAP1B, which shares 3'-untranslated sequence, but not coding sequence, with claustrin. We call this partial cDNA 3'-MAP1B-related clone (3'-MRC), since it is homologous to the 3'-region of the mouse MAP1B sequence. We show by Northern analysis that distinct mRNAs are recognized by the claustrin and 3'-MRC cDNAs, and by RT-PCR that mRNAs encoding these distinct MAP1B-related molecules are present in embryonic chick brain and cardiac and smooth muscle. Our data also suggest a higher level of expression of claustrin mRNA in astrocyte cultures, when compared to 3'-MRC. Our data therefore provide new evidence that alternatively spliced variants of MAP1B are expressed in brain, and that at least one of these variants encodes the claustrin proteoglycan.

Axonal transport of glycoproteins in regenerating olfactory nerve: enhanced glycopeptide concanavalin A-binding.

The size and concanavalin A-binding characteristics of glycopeptides derived from axonally transported glycoproteins were studied in regenerating garfish olfactory nerve. A regeneration related increase was observed in the proportion of total glycopeptide radioactivity associated with the lower molecular weight, dialyzable fraction. There was also a 3-5 fold increase in the axonal transport of low molecular weight concanavalin. A-binding glycopeptides in regenerating nerve. These results suggest a shift to an enhanced synthesis and axonal transport of glycoproteins containing low molecular weight, concanavalin A-binding carbohydrate chains in regenerating nerve.

Monoclonal antibodies specific for ganglion cells in the embryonic chicken neural retina.

Monoclonal antibodies have been produced which recognize antigens on the surface of embryonic chicken neural retina cells. Two monoclonal antibodies, obtained from separate fusions, have been shown by fluorescence-activated cell-sorter analysis to bind to antigens on a subpopulation of neural retina cells. Immunohistochemical methods demonstrate that both monoclonal antibodies bind to the nerve fiber and inner plexiform layers of the embryonic neural retina. These monoclonal antibodies also bind to cell bodies and neurites of cultured retinal neurons and therefore selectively recognize antigens localized to the surface ganglion cells. One of the monoclonal antibodies binds to a 260 kdalton polypeptide, which has been shown to be developmentally regulated by immunoblotting and immunocytochemistry, as it is not detected in late embryonic neural retina. The second monoclonal antibody appears to bind to different antigens in neural and non-neural tissue. In liver extracts the monoclonal antibody recognizes a trypsin-sensitive antigen, but the antibody reacts with trypsin-resistant molecules in neural tissue. This monoclonal antibody may therefore bind a glycolipid component in the neural retina, although this remains to be determined. These antibodies can be used to obtain cell populations highly enriched in retinal ganglion cells.

Identification of sub-populations of chick neural retinal cells by monoclonal antibodies: a fluorescence activated cell sorter screening technique.

An assay was developed which identifies monoclonal antibodies recognizing the cell surfaces of sub-populations of chick retinal cells which survive in culture. Antibodies from hybridoma culture supernatants were bound to monolayers of retina cells followed by a fluorescent secondary antibody. The quantitative fluorescence analysis ability of the fluorescence-activated cell sorter (FACS) was used to determine the fluorescence intensity associated with viable single cells and the frequency with which these cells appear in the total population. Hybridomas were generated which define both overlapping and non-overlapping retina cell populations. The cytotoxic activity of many of the monoclonal antibodies was determined on the FACS by propidium iodide exclusion, and the identity of various sub-populations was demonstrated by immunohistochemical staining of retina sections.

Expression of the barrier-associated proteins EAP-300 and claustrin in the developing central nervous system.

Immunohistochemistry of embryonic chick central nervous system (CNS) and immunocytochemistry of retinal cells were performed to compare and map the expression of two barrier-associated molecules. EAP-300 (embryonic avian polypeptide of 300 kDa) and claustrin (a 320 kDa extracellular matrix keratan sulfate proteoglycan) were both transiently expressed in CNS regions that are considered non-permissive to either neuron migration or axon growth. In the developing spinal cord, EAP-300 and claustrin were both expressed by the marginal zone early in development and by the roof plate later in embryogenesis. In the developing rhombencephalon, immunoreactivity for both molecules was also observed first in the marginal zone, and later expression was restricted mostly to the midline. In the mesencephalon, EAP-300 and claustrin were also localized to the midline, and this expression represented a continuation of the expression observed in the spinal cord roof plate and hindbrain ventral midline. In the developing retina and cerebellum, EAP-300 and claustrin were differentially expressed. In retina, EAP-300 and claustrin were expressed by Müller cells and the optic fiber layer, respectively. In cerebellum at embryonic day 12 (E12), EAP-300 was expressed by Bergman glia, but claustrin was not expressed until E15. Immunocytochemical staining of retinal and cerebellar cultures indicated that EAP-300 was expressed by a subset of radial astrocytes, as confirmed by double labeling experiments with a specific marker for radial astrocytes. These data indicate that in the absence of claustrin expression, EAP-300 was expressed specifically by radial astrocytes during developmental periods of neuron migration. Also, the coexpression of EAP-300 and claustrin in CNS regions considered to be non-permissive to neurite extension suggests that these two developmentally regulated proteins may be associated with barrier function in the developing CNS.

Immunocytochemical localization of a novel radial glial intermediate filament protein.

We have examined by immunocytochemistry the subcellular localization of a chick radial glial protein, named transitin, that by molecular cloning has been shown to be a novel member of the intermediate filament protein superfamily. In astrocytes cultured from E10 chick brain, transitin is localized to the intermediate filament network in accordance with its structural properties. Using confocal microscopy we examined the expression of transitin, vimentin and glial fibrillary acidic protein (GFAP) in cultured astrocytes, and show that transitin co-distributes with these other glial intermediate filament proteins. The expression of transitin, vimentin and GFAP was also compared in embryonic chick spinal cord and brain radial glia, with these studies showing that these intermediate filament proteins display distinct expression patterns during CNS development. Of particular note is the absence of vimentin and GFAP in spinal cord midline radial glia that express transitin protein, and a transient expression of transitin in brain midline radial glia that continue to express vimentin. Our studies presented here therefore indicate that transitin, a novel radial glial intermediate filament protein, may have functions that are unrelated to GFAP or vimentin during CNS development, since transitin is localized to the processes of midline radial glia and is transiently expressed during chick CNS development.

Distribution of the novel developmentally-regulated protein EAP-300 in the embryonic chick nervous system.

In a previous study we described a 300 kDa, developmentally regulated protein identified in embryonic chick neural retina with a monoclonal antibody. Because this protein has been shown to be undetectable in the adult nervous system, and the monoclonal antibody is species-specific, the protein has been named embryonal avian polypeptide of 300 kDa (EAP-300). In the present study we have analyzed the histological expression of EAP-300 during chick embryogenesis. In the developing nervous system, EAP-300 expression was detected as early as Stage 5 (19 h), and was subsequently down-regulated to undetectable levels in the adult. Of particular interest was the association of EAP-300 with putative barriers of neuronal growth, such as the telencephalon/diencephalon glial knot, the dorsal midline in the mesencephalon and the midline in myelencephalon, and the spinal cord roof plate. EAP-300 was also shown to be expressed by Bergmann glia during the period of granule cell migration in the cerebellum. The expression of EAP-300 by radial astrocytes was confirmed in culture by immunofluorescent co-labeling with a MAb to EAP-300 and the R5 MAb, which is a radial astrocyte-specific marker. It has also been shown that EAP-300, when immunopurified from embryonic brain under non-dissociating conditions, co-purifies with a neural keratan sulfate proteoglycan that is also associated with CNS barrier structures during brain development. The restricted expression of EAP-300 in nervous tissue, particularly in CNS barrier structures, suggests that EAP-300 may play an important, but transient, role in the development of the chick nervous system.

Molecular characterization of EAP-300: a high molecular weight, embryonic polypeptide containing an amino acid repeat comprised of multiple leucine-zipper motifs.

In this study we report the biochemical and initial molecular characterization of EAP-300, a developmentally regulated embryonal protein that has been shown previously to be expressed by radial glia in various regions of the CNS, including putative glial barriers. In the present study we have shown that the 300 kDa EAP-300 polypeptide is developmentally regulated in all tissues expressing the protein, which include various PNS and CNS tissues and muscle. In neural tissue the protein is readily detected during early embryogenesis, subsequently down-regulated at later stages, and is not detected in the adult. In contrast to neural tissue, small amounts of the protein are expressed in heart, consistent with earlier studies which showed that EAP-300 expression was maintained in the Purkinje cells of the heart conduction system. Metabolic labeling demonstrates that EAP-300 is a phosphoprotein, and is fatty acylated based on incorporation of [3H]palmitate. We also show that the normal developmental down-regulation of EAP-300 by glia does not occur in vitro, and these data therefore suggest that the signal(s) that regulates EAP-300 gene expression during development in vivo is absent in dissociated cell cultures. We have also initiated molecular studies of EAP-300 by screening embryonic brain cDNA expression libraries with a mixture of EAP-300 monoclonal antibodies. Sequence analysis of partial EAP-300 cDNAs indicate that the protein is related, if not identical, to IFAPa-400, a developmentally regulated intermediate filament-associated protein in chick that is proposed to participate in cell differentiation. These studies also indicate that EAP-300 mRNA is developmentally regulated and is expressed by glial cells in putative CNS barrier structures. Our studies also suggest that two pools of EAP-300 may exist in cells, implying that unlike IFAPa-400 the EAP-300 protein may not always be associated with intermediate filaments. Interestingly, our studies demonstrate that EAP-300 contains a novel repeat amino acid domain comprised of multiple leucine-zipper motifs, which may contribute to its function during glial differentiation.

The developmentally regulated avian protein IFAPa-400 is transitin.

Transitin and IFAPa-400 are developmentally regulated high M(r) proteins expressed transiently in early chick embryogenesis. Both are associated with radially oriented fibers in the developing CNS and with various neural and myogenic tissues before their down-regulation at later stages. Previous studies have shown that IFAPa-400 colocalized and copurified with intermediate filament proteins and recent molecular cloning has indicated that transitin is a member of this family of cytoskeletal proteins. Here, we provide evidence that IFAPa-400 and transitin are the same protein. The sequence of a composite cDNA corresponding to more than 700 amino acids of IFAPa-400 carboxy-terminal extremity is identical to that of transitin. Both proteins exhibit identical apparent M(r) and isoelectric point. Immunopurified IFAPa-400 reacts with different antibodies to transitin and vice-versa. The patterns of expression of both proteins show a perfect coincidence at the tissue level. At the subcellular level, most antibodies to IFAPa-400/transitin decorate a typical intermediate filament network. However, monoclonal antibody A2B11, at the origin of transitin identification, exhibits a staining more typical of a cortical component, suggesting that different populations of transitin exist within the cell.