摘要:

AbstractIn this paper we have described the organization of F‐actin and actin‐binding proteins (ABP): α‐actinin, myosin, tropomyosin, caldesmon, vinculin, talin, and spectrin, in differentiating astroglia in colony cultures. We observed that the microfilament (MF) network arrangements differ at various stages of astroglia development, but the composition of MF bundles and stress fibers is the same at all developmental stages. F‐actin is closely colocalized with myosin, tropomyosin, caldesmon, and α‐actinin. The striated pattern of myosin, tropomyosin, and caldesmon are superimposable. Tropomyosin and caldesmon extend along F‐actin but are interrupted for short periods, whereas myosin is interrupted for longer periods. α‐actinin colocalizes with tropomyosin and caldesmon but not with myosin. In astroglia at different stages of development spectrin is arranged in the form of fine networks spreading through the cell and does not follow the arrangement of MF bundles. Only F‐actin, α‐actinin, and vinculin can be detected at cell‐cell junctions. In the areas of the focal contacts, F‐actin, α‐actinin, vinculin, and talin are present. They overlap each other, although talin and vinculin extend toward the cell membrane beyond F‐actin and α‐actinin.Astroglia undergo well‐defined states of nonmotility, motility, and nonmotility again during differentiation. The changes in motility are paralled by changes in the organization of F‐actin and ABP: as GFAP‐containing intermediate filaments increase in differentiating astroglia, the F‐actin and ABP ar

ISSN:0360-4012    

DOI:10.1002/jnr.490300103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractA specific antiserum against actin‐depolymerizing factor (ADF) was used in a quantitative and immunocytochemical study of ADF in the cerebellum of developing rats. The Triton‐soluble ADF concentration remained stable throughout development. Light and electron microscopic immunocytochemistry showed that ADF was not detected in all cerebellar cells. ADF immunoreactivity was found in Purkinje cells, but not in granule cells. It was found in the Bergmann astrocytes and the astrocytes of the white matter, but not in the oligodendrocytes. The cell bodies and dendrites of Purkinje cells were immunoreactive for ADF but the axons were not. In contrast, the other axons of the white matter (mossy and climbing fibres) were labeled. Thus, ADF was not restricted to either the dendritic or axonal compartments. However, dendritic spines and postsynaptic densities were immunoreactive, whereas presynaptic varicosities were unlabeled. The immunoreactivities for ADF and actin were compared. ADF staining was uniformly distributed throughout the entire dendritic arborization of the Purkinje cell, while filamentous actin is highly concentrated in the dendritic spines, indicating that ADF activity might vary according to its cellular localizat

ISSN:0360-4012    

DOI:10.1002/jnr.490300104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThyroid hormone significantly affects molecular and neuroanatomical properties of the developing nervous system. Altered connectivity in hypothyroidism may reflect reductions in process growth, alterations in process maintenance, or changes in synaptogenesis or synaptic maintenance. These events are dependent on microtubules, neurofilaments, microfilaments, and associated molecular components. Reductions in delivery of microtubules and neurofilaments to the distal axon by slow component a (SCa) of axonal transport may contribute to the neuroanatomical abnormalities of hypothyroidism (Stein et al.,J NeurosciRes 28:121–133, 1991). However, hypothyroidism might also affect the axon and synaptic connections by altering slow component b (SCb), which includes actin microfilaments and proteins that contribute to synaptic function, i.e., clathrin, HSC70 (clathrin uncoating ATPase), spectrin, and calmodulin. To determine the effect of hypothyroidism on SCb proteins, slow axonal transport was analyzed in optic nerves ofhyt/hythypothyroid mice, which have severe primary hypothyroidism, and euthyroid control mice. Clathrin, spectrin, HSC70, and actin showed significant reductions in transport velocity inhyt/hytoptic nerves relative to euthyroid nerves, but the transport rate for calmodulin was less affected. However, the amount of calmodulin was significantly elevated inhyt/hytnerve over euthyroid nerves. Hypothyroidism selectively reduces transport of SCb proteins, which are thought to play significant roles in synaptic function and in the growth cone. The effects of hypothyroidism on microtubules and neurofilaments combined with actions on SCb suggest that changes in neuronal function associated with reduced thyroid hormone during development and maturity (i.e., alterations in neuronal connectivity, nerve conduction, and synaptic function) may be mediated in part by effects on slow axonal transpor

ISSN:0360-4012    

DOI:10.1002/jnr.490300105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractIn the course of screening a rabbit brain cDNA library with a probe for the H neurofilament protein, we identified a neurofilament L‐cDNA. Its nucleotide sequence is 88% identical to that of human, indicating that L is highly conserved among species. The similarities between the sequences of L from rabbit and mouse suggest that the species‐specific accumulation of neurofilaments that occurs in rabbit during aluminum intoxication is not a consequence of the primary structure o

ISSN:0360-4012    

DOI:10.1002/jnr.490300106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C‐terminal “tail” sequences of the two high molecular weight neurofilament sub‐units, NF‐M and NF‐H. A series of recombinant fusion proteins containing selected NF‐M and NF‐H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS‐PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF‐M and NF‐H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF‐M and NF‐H sequence. These antibody studies showed that the extreme C‐termini of NF‐M and NF‐H are immunologically absolutely distinct from one another and also indicate that the extreme C‐terminus of NF‐M is immunologically much more conserved than the analogous region of NF‐H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C‐terminus of NF‐M. We also show that the unusual immunological properties of the bovine NF‐M tail we have previously observed do not extend to the extreme C‐terminal region, which appears immunologically no different from the analogous region of other NF‐M molecules. The peculiarities of bovine NF‐M could be explained by the presence of

ISSN:0360-4012    

DOI:10.1002/jnr.490300107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractWe have inserted aNot1‐Sal1 fragment of the mouse gene coding for the neurofilament protein NF‐H behind the dexamethasone‐inducible transcription promoter of MMTV in a vector derived from pMAMneo(Clonetech Labs). This construct, which includes all four exons of the NF‐H gene, was amplified and in corporated into liposomes for transfection of L cells. Transfectants were selected in G418‐containing medium and cloned. Clones were grown in serum‐containing medium and screened for expression of the NF‐H mRNA by extraction of total RNA, generation of cDNAs by reverse transcription, and amplification of a 900‐base portion of the NF‐H cDNA by PCR. Positive clones were detected by the presence of a band of the correct size on agarose gels. This was confirmed by Southern blotting of the gels probed with a 185‐base segment of the amplified region. Immunofluorescent analysis of two positive clones, C33 and C34, showed that C33 cells grown in serum‐containing medium or in serum‐free medium in the presence of dexamethasone have a network of SMI32 (Sternberger/Meyer Inc.– monoclonal antibody against a nonphosphorylated epitope on NF‐H)‐positive filaments with the same distribution as filaments stained with antibodies to vimentin, while C34 cells do not react with antibodies against neurofilament proteins. Neither clone reacted with antibodies against highl

ISSN:0360-4012    

DOI:10.1002/jnr.490300108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractGlial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. The inhibition or delay in GFAP synthesis might delay scar formation resulting from an insult such as spinal cord injury or central nervous system (CNS) demyelination. The delay in the formation of a physical barrier might allow the neurons and oligodendrocytes to reestablish a functional environment. We delivered antisense GFAP RNA complexed with LipofectinTM(LF), a cationic liposome, into cerebral astrocytes in culture and tested the feasibility of inhibiting GFAP synthesis. Our results demonstrate that LF facilitated antisense RNA uptake into astrocytes. Astrocytes took up3H‐antisense GFAP RNA alone and reached an equilibrium of 7–8.8 ηg per mg protein after 2.5 hr. When complexed with LF, astrocytes could increase the uptake to 14 ηg per mg protein and the time for reaching this quantity was shortened to 10 min. This uptake level was further enhanced if experiments were carried out in HEPES buffered saline (HBS). All uptake studies were dose‐ and time‐dependent. Dibutyryl cyclic AMP (dBcAMP) is known to induce an increase of GFAP content in cultured astrocytes. We studied the effect of LF/antisense GFAP RNA on the GFAP content in dBcAMP (0.25 mM)‐treated astrocytes. Cultures of astrocytes treated with dBcAMP contained almost twice as much GFAP as untreated cultures after 2 days. Similar cultures treated with LF/antisense RNA in HBS did not show an increase but a 30–40% decrease in GFAP content 2 days after treatment. A similar decrease in GFAP content was obtained in cultures grown in a chemically defined medium, another condition known to induce an increase in GFAP content in cultures. This study has demonstrated that antisense GFAP RNA can inhibit GFAP synthesis in astrocytes and may be useful for regulating astrogliosis immediately followi

ISSN:0360-4012    

DOI:10.1002/jnr.490300109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractMammalian neurons and neuron‐like cultured cells express the neural intermediate filament (IF) proteins neurofilament (NF)‐L, NF‐M, NF‐H, and peripherin. To determine whether these proteins are found within the same 10‐nm filament, light and electron microscope immunocytochemistry using peripherin and NF‐specific antibodies was performed on PC12 cells, nervous tissue, and isolated neural filaments from the cauda equina. Double‐label immunofluorescence showed that peripherin and NF‐L, ‐M, and ‐H were found in identical filamentous patterns in interphase and mitotic PC12 cells. Furthermore, expression of mutant peripherin in PC12 cells disrupted not only the peripherin network but also NF‐containing filaments. Immunoelectron microscopy of PC12 cell cytoskeletons showed that peripherin and NF subunit proteins were found in the same filament. In situ, in the sciatic nerve, peripherin/NF‐L or peripherin/NF‐M/‐H double‐label immunofluorescence illustrates at least three types of nerve fibers: those containing NF only, those labeled predominantly for peripherin, and fibers in which peripherin and NF subunits were colocalized. Immunoelectron microscopy of filaments isolated from nerve roots comprising the sciatic nerve also showed the same three labeling patterns seen by light microscopy. Some neural IF appear to contain predominantly NF proteins or peripherin, but in others, both proteins

ISSN:0360-4012    

DOI:10.1002/jnr.490300110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractDouble immunofluorescence studies using antibodies against NF‐L and peripherin revealed three distinct subpopulations of neurons in rat dorsal root ganglia (DRG). In the adult rat, 46% of the DRG neurons were small and peripherin‐positive (NF‐L‐negative), and 48% were large and NF‐L‐positive (peripherin‐negative). About 6% were both peripherin‐ and NF‐L‐positive. All of the DRG neurons reacted with antibodies to NF‐M and nonphosphorylation‐dependent or phosphorylation‐independent antibodies to NF‐H. The neuropeptides were predominantly found in the peripherin‐positive small cell population. Eighty‐seven percent of the peripherin‐positive small cell population contained substance P immunoreactivity, while 43% of this cell population contained CGRP. In contrast, only 18–24% of the NF‐L‐positive large‐cell population contained neuropeptides, and these were primarily in a smaller sized subpopulation. Similar patterns of antigen representation were observed in neonatal (PN2) DRG cell populations. Tissue cultures of sensory ganglion cells from PN2 DRG, in serum‐free medium, stably maintained exclusively peripherin‐positive neurons, with about 5% of these containing coexistent NF‐L immunoreactivity. Very high levels of neuropeptide gene expression were

ISSN:0360-4012    

DOI:10.1002/jnr.490300111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractA 160‐kD polypeptide, which is recognized by an affinity‐purified polyclonal antibody to the 55‐kD tektin‐A polypeptide from sea urchin sperm flagellar microtubules, is associated with neurofilaments in embryonic chick nerve cells. Antibodies to tektin‐A and monoclonal antibodies to the neurofilament triplet proteins colocalize to filaments in cultured nerve cells and to filaments in extracts of chick spinal cord, using indirect immunofluorescence microscopy and immunogold electron microscopy. The antigen reacting with anti‐tektin‐A in chick brain and spinal cord extracts has been identified as a 160‐kD polypeptide by SDS‐PAGE and has been shown to be distinct from the known neurofilament‐triplet proteins by two‐dimensional immunoblot analysis. These data suggest that a unique protein with limited sequence homology to tektin‐A is a component of the neuronal cytoskeleton and is incorporated into or associa

ISSN:0360-4012    

DOI:10.1002/jnr.490300112

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY