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At least 865 records · Page 48Linked to original sources
PubMed1988

Aggregation of intermediate filaments by 2,5-hexanedione: comparison of effects on neurofilaments, GFAP-filaments and vimentin-filaments in dissociated cultures of mouse spinal cord-dorsal root ganglia.

The temporal evolution of changes in the distribution of neurofilaments, vimentin-intermediate filaments (IF) and glial fibrillary acidic protein (GFAP)-IF induced by 2,5-hexanedione (2,5HD) has been investigated in dissociated cultures of fetal mouse spinal cord and dorsal root ganglia (DRG). Aggregates of GFAP-IF, labelled by antibodies against GFAP, appeared in astrocytes approximately one week after axonal swellings filled with neurofilaments were detected in neurons by labelling with antibodies to 68 kD or 200 kD neurofilament proteins. The threshold concentration required for aggregation of GFAP-IF was 1.4 to 2 times that required for neurofilament accumulations. In contrast to findings in cultured human skin fibroblasts, aggregates of vimentin-IF were not found in 2,5HD-treated cultures from mouse tissue (non-neuronal cells of spinal cord-DRG or fibroblasts from skin or muscle). The IF of 3T3 cells, a mouse fibroblast cell line, formed diffuse juxtanuclear aggregates only after high levels of exposure to 2,5HD. The sensitivity of vimentin-IF to aggregation by 2,5HD was proportional to the IF content of the cell type. These differences in the sensitivity of IF to aggregation by 2,5HD among cell types and species explain why IF-aggregates have not been observed previously in non-neuronal cells in human patients with the neuropathy associated with 2,5HD, or in experimental studies with rodents.

Animals↗
PubMed1983

Intermediate filament protein synthesis in preimplantation murine embryos.

The synthesis of two extraembryonic endodermal cytoskeletal proteins (Endo B, Mr = 50,000; Endo A, Mr = 55,000) was detected by immunoprecipitation at the 4- to 8-cell stage of preimplantation mouse development. The first detectable synthesis of both proteins occurs at about the same time as the earliest allocation of cells to the trophectodermal lineage. Both Endo A and B were identified in the two-dimensional gel pattern of blastocyst cytoskeletal proteins prepared by nonionic detergent and high-salt extraction. Endo A and B were identified as the y and x blastocyst cytoskeletal proteins, respectively, previously described by other investigators. Antibodies to Endo B are shown to react with intermediate filaments at the electron microscopic level, confirming that Endo B is an authentic intermediate filament protein. Previously, the TROMA 1 monoclonal antibody prepared by other investigators was shown to react specifically with Endo A and to decorate trophoblast cytoskeletons but did not react with the inner cell mass of blastocysts. Endo B antibodies are now also shown to decorate trophoblast cytoskeletons.

Animals↗
PubMed1989

[Giant axonal neuropathy: intermediate filament disease with involvement of the peripheral and central nervous system].

We report a case of giant axonal neuropathy in a 14 year-old turkish boy with progressive chronic neuropathy and central involvement with mental retardation. CT showed a low density and MRI imaging multiple cavities and hypersignals of the white matter. Nerve and skin biopsies revealed an accumulation of neurofilaments in axonal swellings and an accumulation of intermediate filaments in fibroblasts, Schwann cells, endothelial cells. These findings are in accordance with the reported cases. Giant axonal neuropathy results from a generalized disorder of the intermediate filaments, but the precise biochemical defect is unknown. We would agree with Maia (1988) to name this affection "Giant Axonal Disease".

Axons↗
PubMed1987

[Reorganization of the system of intermediate filaments and detection of the organization centers after centrifugation of cells attached to a substrate].

Cultured pig kidney epithelial cells were centrifuged at 20,000 gav so that the centrifugation force was oriented parallel to the substrate, fixed and processed for indirect immunofluorescent staining with tubulin and vimentin antibodies. After a 2 hour centrifugation vimentin filaments aggregated in the centripetal parts of the cells (probably, because of their association with floating lipid vesicles). Microtubule-organizing centers were found near the centripetal poles of the nuclei, which migrated in the direction of the centrifugal force. The distribution of the cytoplasmic microtubules did not change during centrifugation. The staining of the cultures one hour after centrifugation revealed vimentin-containing spots with radiating intermediate filaments in most of the cells. These spots were localized near the cell nuclei; double immunofluorescent staining with tubulin and vimentin antibodies showed that their position was identical to that of the microtubule-organizing centers. Similar foci of vimentin filaments were seen in the cells after a 3-4 hour centrifugation. Probably, these structures participate in organizing the intermediate filament cytoskeleton in cells.

Animals↗
PubMedDate not supplied

Rearrangements of the intermediate filament GFAP in primary human schwannoma cells.

Loss of the tumor suppressor protein merlin causes a variety of benign tumors such as schwannomas, meningiomas, and gliomas in man. We previously reported primary human schwannoma cells to show enhanced integrin-dependent adhesion and a hyperactivation of the small RhoGTPase Rac1. Here we show that the main intermediate filament protein of Schwann cells, the glial fibrillary acidic protein, is collapsed to the perinuclear region instead of being well-spread from the nucleus to the cell periphery. This cytoskeletal reorganization is accompanied by changes in cell shape and increased cell motility. Moreover, we report tyrosine phosphorylation to be enhanced in schwannoma cells, already described earlier in intermediate filament breakdown. Thus, we believe that Rac activation via tyrosine kinase stimulation leads to GFAP collapse in human schwannoma cells, and suggest that this process plays an important role in vivo where schwannoma cells become motile, unspecifically ensheathing extracellular matrix and forming pseudomesaxons.

Cell Movement↗
PubMed1989

Inhibition of endogenous peroxidase for the immunocytochemical demonstration of intermediate filament proteins (IFP).

The necessity for minimally fixed and processed cell and tissue preparations for immunocytochemical studies of sensitive antigens such as lymphocyte surface markers is well recognised. In order to avoid methanol and hydrogen peroxide, which have been shown to be deleterious for certain antigens, various compounds have been proposed for blocking endogenous peroxidase activity (EPA) in tissue preparations which are to be used in immunoperoxidase reactions. In the present study the deleterious effect of methanol/H2O2 on intermediate filament proteins was demonstrated in both frozen sections and paraffin-embedded tissue. The use of alternative reagents for the non-deleterious blocking of EPA is recommended for immunocytochemical staining with antibodies against intermediate filaments.

Cerebellum↗
PubMed1989

A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus.

We have developed a whole-mount immunocytochemical method for Xenopus and used it to map the expression of the intermediate filament protein vimentin during early embryogenesis. We used two monoclonal antibodies, 14h7 and RV202. Both label vimentin filaments in Xenopus A6 cells, RV202 reacts specifically with vimentin (Mr, 55 x 10(3] on Western blots of A6 cells and embryos. 14h7 reacts with vimentin and a second, insoluble polypeptide of 57 x 10(3) Mr found in A6 cells. The 57 x 10(3) Mr polypeptide appears to be an intermediate filament protein immunochemically related to vimentin. In the whole-mount embryo, we first found vimentin at the time of neural tube closure (stage 19) in cells located at the lateral margins of the neural tube. By stage 26, these cells, which are presumably radial glia, are present along the entire length of the neural tube and in the tail bud. Cells in the optic vesicles express vimentin by stage 24. Vimentin-expressing mesenchymal cells appear on the surface of the somites at stage 22/23; these cells appear first on anterior somites and on progressively more posterior somites as development continues. Beginning at stage 24, vimentin appears in mesenchymal cells located ventral to the somites and associated with the pronephric ducts; these ventral cells first appear below the anterior somites and later appear below more posterior somites. The dorsal fin mesenchyme expresses vimentin at stage 26. In the head, both mesodermally-derived and neural-crest-derived mesenchymal tissues express vimentin by stage 26. These include the mesenchyme of the branchial arches, the mandibular arch, the corneal epithelium, the eye, the meninges and mesenchyme surrounding the otic vesicle. By stage 33, vimentin-expressing mesenchymal cells are present in the pericardial cavity and line the vitelline veins. Vimentin expression appears to be a marker for the differentiation of a subset of central nervous system cells and of head and body mesenchyme in the early Xenopus embryo.

Animals↗
PubMed1984

Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension.

The sequence of the amino-terminal 436 residues of porcine neurofilament component NF-M (apparent mol. wt. in gel electrophoresis 160 kd), one of the two high mol. wt. components of mammalian neurofilaments, reveals the typical structural organization of an intermediate filament (IF) protein of the non-epithelial type. A non-alpha-helical arginine-rich headpiece with multiple beta-turns (residues 1-98) precedes a highly alpha-helical rod domain able to form double-stranded coiled-coils (residues 99-412) and a non-alpha-helical tailpiece array starting at residue 413. All extra mass of NF-M forms, as a carboxy-terminal tailpiece extension of approximately 500 residues, an autonomous domain of unique composition. Limited sequence data in the amino-terminal region of this domain document a lysine- and particularly glutamic acid-rich array somewhat reminiscent of the much shorter tailpiece extension of NF-L (apparent mol. wt. 68 kd), the major neurofilament protein. NF-M is therefore a true intermediate filament protein co-polymerized with NF-L via presumptive coiled-coil type interactions and not a peripherally bound associated protein of a filament backbone built exclusively from NF-L. Along the structurally conserved coiled-coil domains the two neurofilament proteins show only approximately 65% sequence identity, a value similar to that seen when NF-L and NF-M are compared with mesenchymal vimentin. The highly charged and acidic tailpiece extensions of all triplet proteins particularly rich in glutamic acid seem unique to the neurofilament type of IFs. They could form extra-filamentous scaffolds suitable for interactions with other neuronal components.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Application of antibodies to intermediate filament proteins as tissue-specific probes in the flow cytometric analysis of complex tumors.

The flow cytometric (FCM) analysis of carcinomas is often hampered by the presence of stromal and inflammatory cells in the cell suspensions obtained from such neoplasms. Therefore, an FCM method was developed to distinguish epithelial from nonepithelial cells by using polyclonal and monoclonal antibodies to (cyto)keratins, the epithelial type of intermediate filament proteins. Using a model system of cultured bladder carcinoma (T24) and leukemia (MOLT-4) cells, we tested our hypothesis and procedures by labeling cell mixtures with these antibodies. After incubation with an appropriate intermediate filament antibody and propidium iodide staining, the DNA content and distribution of T24 cells could be analyzed separately from MOLT-4 cells. When applied to cell suspensions of endometrial carcinomas, bladder carcinomas and Grawitz tumors, only the epithelial (primarily carcinoma) cells were stained for cytokeratin; these cells could thus be analyzed separately from stromal, inflammatory and other nonepithelial cells. In this way, a more accurate FCM analysis of the malignant fraction within a tumor can be achieved.

Adenocarcinoma↗
PubMed1999

The RNA polymerase II core subunit 11 interacts with keratin 19, a component of the intermediate filament proteins.

We have previously cloned the human RNA polymerase II subunit 11, as a doxorubicin sensitive gene product. We suggested multiple tasks for this subunit, including structural and regulatory roles. With the aim to clarify the human RNA polymerase II subunit 11 function, we have identified its interacting protein partners using the yeast two-hybrid system. Here, we show that human RNA polymerase II subunit 11 specifically binds keratin 19, a component of the intermediate filament protein family, which is expressed in a tissue and differentiation-specific manner. In particular, keratin 19 is a part of the nuclear matrix intermediate filaments. We provide evidence that human RNA polymerase II subunit 11 interacts with keratin 19 via its N-terminal alpha motif, the same motif necessary for its interaction with the human RNA polymerase II core subunit 3. We found that keratin 19 contains two putative leucine zipper domains sharing peculiar homology with the alpha motif of human RNA polymerase II subunit 3. Finally, we demonstrate that keratin 19 can compete for binding human RNA polymerase II subunit 11/human RNA polymerase II subunit 3 in vitro, suggesting a possible regulatory role for this molecule in RNA polymerase II assembly/activity.

Amino Acid Sequence↗
PubMed1989

Intermediate filament proteins immunologically related to desmin in astrocytes: a study of chicken spinal cord by two-dimensional gel electrophoresis and immunoblotting.

Co-migration experiments by two-dimensional SDS-PAGE using chicken spinal cord extracts and desmin purified from chicken gizzard showed that desmin is not present in spinal cord. However, by the immunoblotting procedure, desmin antibodies recognized 3 spinal cord antigens with different molecular weights and isoelectric points than desmin and the glial fibrillary acidic (GFA) protein. These antigens which also reacted with GFA protein antibodies were not identified in chicken gizzard extracts. The reactivity of the antigens with a monoclonal antibody recognizing an epitope common to most intermediate filament proteins (1) suggests that immunostaining of astrocytes with desmin antibodies (2, 3) is due to the presence of new intermediate filament proteins immunologically related to desmin.

Animals↗
PubMed1983

Interaction of frog virus-3 with the cytoskeleton. I. Altered organization of microtubules, intermediate filaments, and microfilaments.

The progressive cytoskeletal alterations of frog virus 3-infected baby hamster kidney (BHK) and fathead minnow (FHM) cells were studied by immunofluorescence and electron microscopy. The virus assembly sites, which contain viral genomes and viral proteins, were detected in the cytoplasm at 4 h (FHM) or 6 h (BHK) and mature virions appeared 2 h later. When infected cells were treated with Triton X-100, the assembly sites were found in association with the cytoskeleton. In infected cells, the number of microtubules progressively decreased but a few microtubules traversing in the vicinity of the assembly sites remained intact. Early in infection, the intermediate filaments retracted from the cell periphery, delimited the forming assembly sites, and remained there throughout infection. We suggest that intermediate filaments are involved in the formation of assembly sites. In addition, the filaments either by themselves or in conjunction with microtubules may anchor the assembly sites near the nucleus. The microfilament bundles (stress fibers) disappeared with the formation of assembly sites, and late in infection many projections containing microfilaments and virus particles appeared at the cell surface. The observation suggests a role for microfilaments in virus release. Taken together, these results provide the first example of a virus-infected cell in which all three cytoskeletal filaments show profound organizational changes and suggest an active participation of the host cytoskeleton in viral functions.

Animals↗
PubMed1982

Intermediate filaments and their interaction with membranes. The desmosome-cytokeratin filament complex and epithelial differentiation.

Intermediate-sized filaments represent a class of morphologically similar but biochemically and immunologically distinguishable cytoplasmic protein polymer structures. Five major filament types have been identified (cytokeratin, vimentin, desmin, neurofilament protein, glia filament protein) and antibodies to these proteins have been used for distinguishing different cell types and tumors derived therefrom. Epithelial and carcinoma cells are characterized by the presence of cytokeratin filaments and desmosomal elements identified by antibodies to certain high molecular weight proteins of desmosomal plaques. However, the specific pattern of cytokeratin polypeptides is different in different epithelia. The potential value of cell type identification by immunological reactions with antibodies to cytoskeletal proteins in tumor diagnosis is discussed.

Animals↗
PubMed1991

Influence of fibric acid derivatives on intermediate filament proteins in myocardiocyte cultures.

We analyzed desmin and vimentin accumulation in chick myocardiocyte cultures treated with the fibric acid derivatives bezafibrate, fenofibrate and gemfibrozil. The most noteworthy finding was the 50% decrease in the cytoplasmic desmin fraction in cells treated with gemfibrozil in comparison to control cultures, and the 19% increase in the cytoskeletal fraction in cultures treated with gemfibrozil and with bezafibrate. Vimentin accumulation by cells treated with bezafibrate was similar to that in control cultures, however the cytoskeletal vimentin fraction rose by 26% after treatment with gemfibrozil, and fell 13% after treatment with fenofibrate. No vimentin was found in the cytoplasmic fraction of cell treated with bezafibrate. Given the role of intermediate filaments in heart muscle contraction, fibric acid derivative- induced changes in the cytoplasmic and cytoskeletal concentrations of intermediate filament proteins may be related with the secondary effects of these drugs on heart rate.

Animals↗
PubMed1984

58,000 dalton intermediate filament proteins of neuronal and nonneuronal origin in the goldfish visual pathway.

A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two-dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal-enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one-dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one-dimensional gel electrophoresis.

Animals↗
PubMed1992

Salt-stable interaction of the amino-terminal head region of vimentin with the alpha-helical rod domain of cytoplasmic intermediate filament proteins and its relevance to protofilament structure and filament formation and stability.

Previous studies have shown that the non-alpha-helical, amino-terminal head region of vimentin is essential for the formation and stability of vimentin intermediate filaments (IFs). In order to specify its target site on companion protein subunits, it was cut off from vimentin at amino acid position 96 with lysine-specific endoproteinase and allowed to react with intact vimentin and other IF proteins. In solution of high salt concentration (500 mM KCl), the isolated polypeptide (vim NT) showed a high affinity for all cytoplasmic IF proteins tested, but not for nuclear lamins. Employing limited digestion of the IF proteins with different proteinases, the binding site was shown to reside in their alpha-helical rod domains. Other polypeptides possessing alpha-helical regions with the potential to form coiled-coil structures like tropomyosin and myosin subfragment 2 did not react with vim NT. The binding to IF proteins was strongly inhibited by phosphorylation of vim NT and totally abolished in the presence of 200 mM arginine hydrochloride, whereas the same concentration of lysine hydrochloride was ineffective. Limited chymotryptic digestion of vim NT produced polypeptides that were unable to react with the alpha-helical region of vimentin at high salt concentration. Consistent with these observations, vim NT strongly inhibited filament formation in vitro from protofilamentous vimentin. A 14-mer oligopeptide comprising the amino acids 3 to 16 of the amino terminus also inhibited filament formation, though to a lesser extent. Conversely, vim NT and, with a lower efficiency, the 14-mer oligopeptide also severely affected the structure of preformed vimentin filaments by unraveling them. Phosphorylated vim NT was considerably less active in this respect. Further digestion of the rod domain of vimentin with chymotrypsin yielded 17.4 and 21 kDa polypeptides, which were tentatively characterized as originating from the carboxy- and amino-terminal half of the rod domain, respectively. Both formed salt-stable complexes with vim NT, the smaller polypeptide with a higher efficiency than the larger one. These results suggest that the staggered, antiparallel arrangement of the two coiled-coils in the protofilaments of IF proteins is, at least in part, determined by the twofold, symmetrical association of the amino-terminal head regions of one coiled-coil rope structure with the carboxy-terminal halves of the alpha-helical rod domains of the other coiled-coil and that similar interactions occur during filament assembly and in the intact filament.

Amino Acid Sequence↗
PubMed1993

Concurrence between the molecular overlap regions in keratin intermediate filaments and the locations of keratin mutations in genodermatoses.

By analysis of the existing available data, we have found that the locations of disease-causing mutations in epidermal keratin genes are distributed in a non-random manner. Most occur in exons 1 and 7 which encode the highly conserved 1A and 2B rod domain sequence regions of the keratin chains. Recent structural studies have suggested these sequences define an important overlap between neighboring molecules in keratin intermediate filaments. In order to better map the extent of these overlap sequences and concurrently to identify those sequences likely to be sensitive to mutations, we have used a series of synthetic peptides in an established filament disassembly assay. Thus residue positions 7-16 of the 1A and positions 107-117 of the 2B rod domain segments describe the extent of the molecular overlap window wherein mutations in keratin (and perhaps other) intermediate filaments are most likely to alter filament stability and lead to abnormalities.

Amino Acid Sequence↗
PubMed1995

Intermediate filaments in cardiac myogenesis: nestin in the developing mouse heart.

By using immunohistology combined with immunoblotting, cell culture, and RT-PCR, we show that the intermediate filament protein nestin is transiently expressed in the midembryonic mouse heart. Monoclonal antibody (MAb) Rat-401, known to react with nestin in neural and skeletal muscle cells, was also found to react with ventricular and atrial cells throughout the mouse heart from embryonic day 9 (E9) through E10.5. Both before (E8.5) and after (E11-adult) this brief period, staining with Rat-401 was absent from atrial and ventricular myocytes. To evaluate the specificity of staining with MAb Rat-401 in the heart, we used immunoblotting, cell culture, and RT-PCR to verify that the authentic nestin protein and mRNA were expressed in cardiomyocytes of the E10 mouse. Nestin expression is the first molecular marker for this distinct midembryonic period of heart development.

Animals↗