Cloning of Zebrafish Neurofilament cDNAs for Plasticin and Gefiltin: Increased mRNA Expression in Ganglion Cells After Optic Nerve Injury

Abstract: During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.     

Organization, Sequence, and Expression of a Gene Encoding Goldfish Neurofilament Medium Protein

Abstract: The goldfish visual pathway displays a remarkable capacity for continuous neurogenesis, plasticity, and regeneration. The intermediate filament protein composition of this system differs from that of higher vertebrates, which lack the capacity for continued nerve growth and development. In an effort to determine how intermediate filament proteins are regulated during nerve growth, we isolated and characterized cDNA and genomic clones representing the goldfish neurofilament medium (NF-M) protein. The tissue-specific expression of goldfish NF-M mRNA was analyzed by RNase protection assays and by in situ hybridization. The expression of goldfish NF-M is qualitatively the same as in other species. Although the intermediate filament protein composition of the goldfish visual pathway is unusual when compared with higher vertebrates, the goldfish NF-M protein is similar to higher vertebrate NF-M proteins. In addition, the organization of the goldfish NF-M gene is identical to the NF-M genes in all other vertebrate species. In contrast, the promoter region of the goldfish NF-M gene has several potential regulatory sequences that are not found in the promoter regions of higher vertebrate NF-M genes.     

Cloning of Multiple Forms of Goldfish Vimentin: Differential Expression in CNS

Abstract: In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal λgt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin α and vimentin β. RNase protection assays indicate that vimentin α mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin β mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin α is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two-dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.     

Identification of a Peripherin Dimer: Changes During Axonal Development and Regeneration of the Rat Sciatic Nerve

Abstract: Western blotting of rat dorsal root ganglion (DRG) and sciatic nerve under nonreducing conditions revealed that a peripherin-specific antibody recognized a protein species of 116/130 kDa, pi 5.6, in addition to peripherin (56 kDa, pl 5.6). We showed that this 116/130 kDa protein is a disulfide dimer of peripherin, because it gave rise to a single protein band comigrating with peripherin under reducing conditions and yielded the same proteolytic pattern as peripherin upon N-chlorosuccinimide digestion. In addition, the immunological characteristics of the resulting peptides were identical to those of peripherin. We investigated the changes in peripherin monomer and dimer protein levels during axonal development and regeneration. During postnatal development, quantitative analysis of western blots of DRG proteins showed a significant increase in peripherin monomer (+52%) and dimer (+33%) levels from the day of birth [postnatal day 0 (PO)] to P7. The monomer levels remained high until P14 and then decreased so that at P21 and later ages, the monomer levels were similar to those observed at birth. In contrast, the dimer levels decreased continuously after P7, and in the adult, its level represented only 30% of the level at birth. Changes in [³⁵S]methionine incorporation into adult DRG proteins were studied during regeneration of axotomized sciatic axons. Quantitative analysis of proteins showed a strong increase in labeling of both peripherin monomer (+56%) and dimer (+88%) 7 days after the crush. These levels, which remained high until 28 days after the axotomy, had returned to normal 70 days post axotomy. Our results show that peripherin monomer and dimer greatly increase during DRG fiber development and regeneration, suggesting that the two forms are involved in the growth of axons.     

Expression of a 48-Kilodalton Growth-Associated Protein in the Goldfish Retina

One of the most striking molecular correlates of optic nerve regeneration in the goldfish is the increased labeling of a 48 kilodalton (kD) acidic protein that is conveyed to the developing nerve endings from the retina by rapid axonal transport. The present study examined the biosynthesis and molecular characteristics of this protein. Retinas derived either from intact controls or from goldfish undergoing optic nerve regeneration (10-14 days postcrush) were pulse-labeled with [3H]proline or [35S]methionine, followed by subcellular fractionation and analysis of protein synthesis patterns by two-dimensional gel electrophoresis and fluorography. Synthesis of the 48-kD acidic protein (termed here GAP-48) was detected only in retinas that were undergoing axonal regeneration. Pulse-chase labeling experiments demonstrated that the protein undergoes a post-translational modification that requires 15-20 min. This processing could be selectively blocked by tunicamycin, an inhibitor of protein N-glycosylation. The protein was also found to incorporate low levels of phosphate in vitro. Thus, the differential appearance of GAP-48 in regenerating axons might be regulated either at the level of gene expression or by selective posttranslational processing in retinal ganglion cells. By the criteria of molecular weight, isoelectric point, anomalous migration properties on sodium dodecyl sulfate-polyacrylamide gels, phosphorylation, subcellular distribution, and the pattern of digestion products generated by Staphylococcus aureus V8 protease, GAP-48 appears to be equivalent to the B-50 (F-1) phosphoprotein of the mammalian brain.     

Analysis and Comparison of In Vitro Synthesized Glial Fibrillary Acidic Protein with Rat CNS Intermediate Filament Proteins

Intermediate filament (IF) proteins from rat spinal cord were analyzed by two-dimensional gel electrophoresis and compared with the in vitro translation products of a messenger RNA-dependent reticulocyte lysate system stimulated with 16-day-old rat brain polysomes. In two dimensions, the molecular weight 49,000 to 50,000 band of the IF preparation resolved to seven spots, whereas antiserum to glial fibrillary acidic (GFA) protein precipitated only two immediately adjacent radiolabeled in vitro synthesized products, with molecular weights of 49,000 to 50,000. Autoradiographs of two-dimensional gels of extracted IF proteins incubated with iodinated IgG fraction of GFA protein antiserum showed that all seven spots were recognized by the antiserum. These observations suggest that the primary gene product of GFA protein is modified either by post-translational processing or experimental artifact.     

Comparison of the glial investment of normal and regenerating fiber bundles in the optic nerve and optic tectum of the goldfish and the Crucian carp

Summary The architecture of normal and regenerating nerve fiber bundles in the optic nerve of the goldfish and the Crucian carp was compared to that of the axonal fascicles in the optic tectum of these teleost species with the use of ultrathin sections and freeze-fracture replicas. The fascicles in the optic nerve are clearly demarcated by astrocytic processes, in contrast to the fascicles in the tectum. No astrocytes could be identified in the tectum; in this region processes of astrocytes or of radial glial cells do not form channeling structures reminiscent of those in the optic nerve. Furthermore, tectal blood vessels lack complete investments of glial processes. It can be assumed that at least in lower vertebrates a framework of astrocytic processes might be important for growth of optic fibers over large distances, i.e., from the eye to the tectum, but may be dispensable in the target region itself.     

Alterations in mRNA Translation Products Associated with Regenerative Responses in the Retina

Translation products of mRNA from retinas of goldfish optic nerve (representing a regenerative CNS) and adult rabbit optic nerve (representing a nonregenerative CNS which can be induced to express regenerative characteristics) were examined by one- and two-dimensional gel electrophoresis. Translation products from retinas of the regenerating goldfish optic nerve included polypeptides barely detectable in the translation products of mRNA derived from retinas of uninjured controls. Some of these polypeptides, of apparent molecular weights 24-28, 43-49, 60, and 65 kilodaltons can be considered as growth-associated polypeptides described in other regenerative and developing systems. The induction of regeneration-associated characteristics in the injured adult rabbit optic nerve, "implanted" with diffusible substances from nonneuronal cells of regenerative or growing nerve, is reflected by changes in the mRNA translation products of the retina. Among such translation products are those of the following molecular weights: 16-18, 28, 32-35, 43-47, and 56-60 kilodaltons, and some higher-molecular-weight species.     

Regeneration of the goldfish retina after exposure to different doses of ouabain

Summary After ouabain-induced degeneration, the retina of the goldfish shows a remarkable regeneration capacity. The extent of the damage depends on the dose of ouabain used in the experiment. After intraocular injection of 7l 10^–5 M ouabain, the ganglion cells and the cells of the inner nuclear layer (INL) become necrotic except for most of the outer horizontal cells, some bipolar cells, and Müller cells. The outer nuclear layer (ONL) and the marginal growth zone at the ora serrata remain intact; the plexiform layers become spongy. The degenerated material is removed by the proliferated reactive macroglial cells and invading macrophages. The degenerated cellular elements of the retina are replaced by mitosis of neuroblasts in the marginal growth zone and of cells in the ONL.After intraocular injection of a 5-fold higher dose of ouabain (7 l 5·10^–5M), the degeneration of the retina proceeds more rapidly and completely. In this experiment, the ONL is destroyed and the receptor outer segments are phagocytosed by cells of the pigment epithelium. In contrast to the regeneration of the amphibian retina, in the goldfish cells of the pigment epithelium do not participate by metaplastic transformation in the regeneration of the retina. The only source of cellular regeneration of the retina after complete destruction of its differentiated neural elements is the marginal growth zone, which is highly resistant to ouabain. The rate of mitoses in this region is strongly increased. The derivatives of these cells spread out tangentially over the entire fundus of the eye in a concentric manner. In this regenerate, mitotic processes continue in a radial direction, resulting in thickening and layering of the new retinal formation.     

Peripherin Is Tyrosine-Phosphorylated at Its Carboxyl-Terminal Tyrosine

Abstract: Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim⁻ cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim⁻ cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim⁻ cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.     

Glial fibrillary acidic protein and desmin in salivary neoplasms

The presence of intermediate filament proteins (IFP) in normal salivary gland tissue and in a number of salivary gland neoplasms has been investigated by immunohistochemical techniques on frozen sections. Cytokeratins (CKs) were seen in almost all normal epithelial cells. In the parotid gland and in palatal gland tissue, a co-expression of cytokeratin and glial fibrillary acidic protein (GFAP) was seen in some myoepithelial cells, but this was not apparent in the submandibular gland. In some pleomorphic adenomas, carcinomas in pleomorphic adenomas, one mucoepidermoid carcinoma, one mucus-producing adenopapillary carcinoma and one adenoid cystic carcinoma, cells expressing three different IFP classes were found (CKs, vimentin, GFAP). These cells were most often situated peripherally in the tumour cords or ducts. The cytokeratin pattern in these cells, as revealed by mAbs PKK1-3, was similar to that in normal myoepithelial cells. Furthermore, reactivity for a fourth class of IFP, desmin, could be seen in this cell type in two carcinomas in pleomorphic adenomas, and also in a few cells in a pleomorphic adenoma and an adenoid cystic carcinoma. Thus the pattern of IFP expression in salivary gland neoplasms, is very complex, and cannot always be related to the normal tissue.     

角蛋白缺陷疾病转基因动物模型的研究进展

角蛋白是哺乳动物角质形成细胞的主要结构蛋白,其基因的正确表达是细胞稳定和功能正常的基础。角蛋白的基因突变可导致一系列的遗传性疾病。近年来,转基因动物模型的建立在疾病的发病机制、基因间的相互关系方面带给了我们全新的视角。本文就角蛋白相关疾病动物模型的研究现状做一综述。     

Syncoilin isoform organization and differential expression in murine striated muscle

Syncoilin is a 64 kDa intermediate filament (IF) protein expressed in myocytes at the sarcolemma, perinucleus, myotendenous and neuromuscular junctions. Here we present a revised domain projection and structural analysis for the original isoform (sync-1) and introduce two novel syncoilin isoforms (sync-2 and sync-3) generated by exon splicing. On the basis of consensus identity we propose that syncoilin be reclassified as a type III IF protein. All three syncoilin isoforms lack a L1 domain, a significant departure from standard IF rod domain projections that is likely to impact significantly on their biological function. Our analyses indicate that syncoilin is unlikely to form classical intermediate filament structures by itself, and that the significant difference in C-terminal structure between the three isoforms indicates that they may play divergent roles in myocytes. We show that despite lacking an apparent structural role in striated muscle, syncoilin isoforms are differentially and strongly upregulated in response to cardiotoxin induced regeneration and denervation induced atrophy in the C57BL/6 mouse, possibly suggesting an atypical role for syncoilin in muscle.     

Coding sequence and flanking regions of the mouse vimentin gene

Summary Using a polyclonal antibody, a cDNA clone coding for part of mouse vimentin was identified in a gt11 expression library. DNA from this clone was used to screen a genomic library from Ehrlich Ascites Tumor cells for the mouse vimentin gene. A clone was found which contained the whole coding sequence and a large part of the 5- and 3-untranslated sequences. It was used to prepare a construct equivalent to a full-length cDNA clone. Extensive homologies to the vimentin sequence from other species were found for the coding and 3-untranslated sequences and the promoter region.     

Immunohistochemical Localization of Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Optic Nerve: Specific Molecular Markers for Optic Nerve Structures

The predominant proteins (58K) of the intermediate filament complex in the goldfish visual pathway consist of a series of isoelectric variants. Previous biochemical studies have shown that proteins ON1 and ON2 are of neuronal origin, whereas ON3 and ON4 are of nonneuronal origin. Polyclonal antibodies, purified by affinity chromatography, that are specific for ON1 and ON2 or ON3 and ON4 have been used to localize histologically the ON proteins within the normal and crushed optic nerve. Anti-ON1/ON2 antiserum presented a pattern consistent with intraaxonal staining. A nonneuronal staining pattern was observed with anti-ON3/ON4 antiserum. The two patterns were distinct from and complementary to each other. The data suggest that ON3 and ON4 represent a novel glial fibrillary acidic protein. The results are discussed in terms of the function of these proteins in development, plasticity, and regeneration.     

Isolation of the intermediate filament protein vimentin by chromatofocusing

A novel, simple and relatively rapid method is described for the isolation of the intermediate-sized filament protein vimentin from eye lens tissue. Chromatofocusing is applied as the sole purification step. The apparent isoelectric point of the protein in 6 M urea and at 22°C is 4.9. Electrophoretic mobility on one- and two-dimensional polyacrylamide gels, solubility in 6 M urea and amino acid composition were used for identification     

Functional and morphological regeneration of olfactory tracts and subtracts in goldfish

Goldfish are ideal vertebrates for the study of regeneration within the central nervous system. The present behavioural and neuroanatomical investigations after bilateral transection of the entire olfactory tracts of either lateral or medial subtracts have been designed (1) to examine the relationship between morphological changes and changes in the perception of spontaneously preferred chemosensory stimuli, (2) to investigate the animals' ability to qualitatively discriminate amino acids in olfactory concentrations (below taste threshold, 10^-6–10^-8 M), one of which had been rewarded preoperatively (specific regeneration), and (3) to examine the discriminative ability for amino acids at concentrations above taste threshold (> 10^-5 M) in intact sham-operated, and in operated specimens at various time intervals before functional regeneration. Within 10–14 days after bilateral transection of the lateral olfactory tracts, specific regeneration was observed. After bilateral transection of the medial olfactory tracts, no immediate behavioural change was recorded for 1 week. Thereafter, goldfish behaviour became unstable and dropped to the chance level for 3–4 weeks. Subsequent to this time the goldfish returned to the preoperative level. Following bilateral crushing of the olfactory tracts and after total tractotomy, a specific regeneration was observed after 4 weeks and 6–8 weeks, respectively, post op. HRP studies showed that after bilateral lesioning a qualitative reinnervation of the respective nuclei within the forebrain by the medial and lateral olfactory subtracts was evident.Abbreviations FB funnel biting - FO funnel orientation - HRP horseradish peroxidase - LOT lateral olfactory tract - MOT medial olfactory tract     

The cephalochordate Branchiostoma genome contains 26 intermediate filament (IF) genes: Implications for evolution of chordate IF proteins

We analyzed the draft genome of the cephalochordate Branchiostoma floridae (B. floridae) for genes encoding intermediate filament (IF) proteins. From 26 identified IF genes 13 were not reported before. Four of the new IF genes belong to the previously established Branchiostoma IF group A, four to the Branchiostoma IF group B, one is homologous to the type II keratin E2 while the remaining four new IF sequences N1 to N4 could not be readily classified in any of the previously established Branchiostoma IF groups. All eleven identified A and B2-type IF genes are located on the same genomic scaffold and arose due to multiple cephalochordate-specific duplications. Another IF gene cluster, identified in the B. floridae genome, contains three keratins (E1, Y1, D1), two keratin-like IF genes (C2, X1), one new IF gene (N1) and one IF unrelated gene, but does not show any similarities to the well defined vertebrate type I or type II keratin gene clusters. In addition, some type III sequence features were documented in the new IF protein N2, which, however, seems to share a common ancestry with the Branchiostoma keratins D1 and two keratin-related genes C. Thus, a few type I and type II keratin genes existed in a common ancestor of cephalochordates and vertebrates, which after separation of these two lineages gave rise to the known complexities of the vertebrate cytoplasmic type I–IV IF proteins, as well as to the multiple keratin and related IF genes in cephalochordates, due to multiple gene duplications, deletions and sequence divergences.