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In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed.

Intact neurofilaments (NF) purified from mammalian brain and spinal cord promote the assembly of microtubules in solutions of pure phosphocellulose (PC)-purified tubulin. This assembly is temperature-dependent and is inhibited by mitotic spindle inhibitors. The ability of NF to induce microtubule formation is 20% of that of purified microtubule-associated proteins (MAPs), whereas MAPs comprise less than 5% of the protein in the NF preparations. The inducing activity of NF is rapidly lost on boiling. When intact NF are incubated with PC-tubulin and then centrifuged, tubulin is sedimented together with the filaments. This association is inhibited by colchicine and podophyllotoxin and is cold-sensitive. NF purified to homogeneity under denaturing conditions and then reassembled completely lack the ability to promote the assembly of PC-tubulin or to bind tubulin on a centrifugation assay. No MAPs are present in these preparations, though these filaments have the ability to bind exogenous MAPs. While these experiments do not rule out an intrinsic microtubule-assembly-promoting activity, they suggest that this activity is due to nontriplet proteins in the preparation, most likely filament-associated MAPs.

Specific interactions between neurons and glia dissociated from early postnatal mouse cerebellar tissue were studied in vitro by indirect immunocytochemical staining with antisera raised against purified glial filament protein, galactocerebroside, and the NILE glycoprotein. Two forms of cells were stained with antisera raised against purified glial filament protein. The first, characterized by a cell body 9 microns diam and processes 130-150 microns long, usually had two to three neurons associated with them and resembled Bergmann glia. The second had a slightly larger cell body with markedly shorter arms among which were nestled several dozen neuronal cells, and resembled astrocytes of the granular layer. Staining with monoclonal antisera raised against purified galactocerebroside revealed the presence of immature oligodendroglia in the cultures. These glial cells constituted approximately 2% of the total cell population in the cultures and, in contrast to astroglia, did not form specific contacts with neurons. Staining with two neuronal markers, antisera raised against purified NILE glycoprotein and tetanus toxin, revealed that most cells associated with presumed astroglia were small neurons (5-8 microns). After 1-2 d in culture, some stained neurons had very fine, short processes. Nearly all of the processes greater than 10-20 micron long were glial in origin. Electron microscopy also demonstrated the presence of two forms of astroglia in the cultures, each with a different organizing influence on cerebellar neurons. Most neurons associated with astroglia were granule neurons, although a few larger neurons sometimes associated with them. Time-lapse video microscopy revealed extensive cell migration (approximately 10 microns/h) along the arms of Bergmann-like astroglia. In contrast, cells did not migrate along the arms of astrocyte-like astroglia, but remained stationary at or near branch points. Growth cone activity, pulsating movements of cell perikarya, and ruffling of the membranes of glial and neuronal processes were also seen.

Following a brief period of heat stress, the two highly conserved mammalian stress proteins, hsp68 and 70, were examined with respect to their intracellular locations. In four independent cell lines, hsp68 and 70 were found to partition into both Triton X-100-soluble and insoluble fractions as assessed by two-dimensional gel analysis of newly synthesized polypeptides, whereas a fifth cell line showed these proteins only in the Triton X-100-insoluble fraction. In addition, a previously described cell fractionation technique was utilized to gain information regarding the segregation of the two major mammalian stress proteins, hsp68 and 70, into distinct biochemically and morphologically characterized subcellular compartments of PtK2-epithelial cells. Two cytoskeletal-specific agents, taxol and colchicine, were also probed for their effects on the disposition of these polypeptides. Under our conditions of acute heat exposure, hsp68, 70 and their isoforms were globally distributed in all subcellular fractions examined, with a few notable exceptions in drug-treated cells. Colchicine, a microtubule-depolymerizing drug, inhibited the association of hsp68 and its variants with the double-detergent-extractable labile "cytoskeleton," whereas taxol, a microtubule-stabilizing agent, in some manner, facilitated the transit of hsp68 and its isovariants from a cytoplasmic to nuclear domain. Degree of cell density is a factor which influences the synthesis of various cytoskeletal proteins; therefore, we studied the effect of cell confluency on the disposition of mammalian stress proteins hsp68 and 70 in human FS-4 fibroblasts. In confluent cultures, where cell-cell contact was maximal, we observed the appearance of a previously undetected polypeptide which was not found in sparsely populated cultures. This protein may represent a post-translationally modified isoform of a preexisting heat shock protein, or perhaps, a novel stress protein.

We have studied the onset of expression of glial filament protein (GFP) in astrocytes along a single axon trajectory, the mouse retinal axon pathway, and the relationship of GFP expression to maturation of astroglial morphology. In fetal optic nerve from embryonic day (E) 12 to E16, primitive glia (neuroepithelial cells) lack GFP, but express vimentin and contain intermediate filaments. GFP is expressed at E17 in two gradients: cells in the optic nerve become GFP-positive first in the borders of the nerve, then in the central nerve by postnatal day (P)0. The second gradient is a distoproximal one, with GFP appearing in the optic nerve at E17, in the optic chiasm by PO, and in the optic tract by P3. The expression of GFP in the optic nerve marks the transformation of radial neuroepithelial cells to multipolar astroglia, accomplished by outgrowth of filament-rich glial processes tipped by a growth cone. Several days after the onset of GFP expression in each portion of the pathway astrocytes exhibit a transient increase in staining, and resemble reactive astrocytes after injury. During this period, filaments are arranged in densely packed bundles, and appear coalesced at points. Thus, primitive glial cells in optic nerve express vimentin. In the retinofugal pathway, GFP is expressed in a distinct spatiotemporal sequence from optic nerve to optic tract. Finally, in contrast to neurons, the extension of astroglial processes is accompanied by the increased expression and assembly of intermediate filaments.

Mammalian neurofilaments prepared from brain and spinal cord by either of two methods partially inhibit the in vitro assembly of microtubules. This inhibition is shown to be due to the association of a complex of high molecular weight microtubule-associated proteins (MAP1 and MAP2) and tubulin with the neurofilament. Further analysis of the association reveals a saturable binding of purified brain MAPs to purified neurofilaments with a Kd of 10(-7) M. Purified astroglial filaments neither inhibit microtubule assembly nor show significant binding of MAPs. It is proposed that the MAPs might function as one element in a network of intraorganellar links in the cytoplasm.

Exposure of PC12 cells to nerve growth factor (NGF) has been shown to induce an mRNA that encodes a novel neuronal intermediate filament protein. The findings presented here concern the identity of this filament protein. The major protein in NGF-treated PC12 cell cytoskeletons derived by extraction with 1% Triton X-100 is of apparent Mr = 58,000, focuses by isoelectric focusing as several closely spaced spots of pl 5.6-5.8, and is elevated relative to non-NGF-treated cells. Partial microsequencing of this material reveals 2 internal sequences that are identical to a 14-residue sequence encoded by the NGF-regulated clone 73 mRNA, but not to sequences of other known proteins. An antiserum raised against a 19-residue synthetic peptide corresponding to the deduced C-terminus of the protein encoded by the NGF-regulated clone 73 mRNA specifically recognizes the 58,000-Mr protein. Properties of the 58-kilodalton protein strongly suggest that it corresponds to an intermediate filament protein (peripherin) previously identified in PC12 cells and in peripheral and certain CNS neurons. Identification of the intermediate filament protein encoded by an NGF-induced message should facilitate studies of its regulation and function.

Morphological and biochemical evidence have suggested that the components of the neuronal cytoskeleton, microtubules and neurofilaments (NF), interact with each other. Microtubule-associated proteins (MAPs) are plausible candidates for mediating some of these interactions and have been shown to bind to neurofilaments, as well as induce the formation of a viscous complex between neurofilaments and microtubules. By binding 32P-labeled MAPs to neurofilament proteins, which were transferred electrophoretically to nitrocellulose, we determined that, of the three neurofilament subunits, only the core NF70 subunit bound MAPs. The binding to electrophoretically transferred NF70 was specific, saturable, and reversible. Binding parameters were estimated by binding 32P-labeled MAPs to purified NF70 immobilized on nitrocellulose. Approximately 1 mol of MAPs bound per 45 +/- 15 mol of NF70 with an approximate Kd approximately 2.0 +/- 0.9 X 10(-7) M (n = 8). Reassembled filaments in suspension were used to confirm the specific binding. Tubulin and NF70 apparently bind to different sites on MAPs.

The hydrogen exchange behavior of a small allosterically responsive set of exchanging hydrogens was studied in hemoglobin A and in some chemically modified hemoglobins. The set experiences an exceptionally large change in exchange rate through hemoglobin's allosteric transition. This indicates, according to the local unfolding model of H-exchange, that a large change in allosteric free energy impinges on the opening segment that exposes these protons to exchange. In oxyhemoglobin the set consists of 5 to 6 protons which exchange with a half-time of 20 s at pH 7.4 and 0 degrees C. In deoxyhemoglobin the set splits into a slower and a faster half. The slower 3 protons exchange more slowly than in oxyhemoglobin by a factor of 5000 (26 h half-time) and are 5-fold slower still in the presence of pyrophosphate or inositol hexaphosphate (136 h half-time). The other 2 to 3 protons exchange about 20-fold faster in both cases (about 2 h and 10 h half-times). The effect of some chemical modifications was tested, including reaction with iodoacetamide and N-ethylmaleimide and cleavage with carboxypeptidases A and B. In all cases the 3 slower protons continue to behave as a cohesive set and in the various modified deoxyhemoglobins their exchange is accelerated by factors ranging between 1 and 3 decades. These factors correlate with the effect of the different modifications on hemoglobin cooperativity.

A rapid batchwise purification procedure for neurofilament proteins from bovine spinal cord is described. A crude filament fraction can be obtained by treating the tissue with Triton X-100, followed by centrifugation through sucrose. From this crude filament fraction the protein is processed through a batch purification procedure using hydroxyapatite in 8 M urea. With this procedure, approximately 0.5 g of purified neurofilament protein is obtained from a single bovine spinal cord in less than 3 days.

Highly purified preparations of bovine brain and rabbit nerve root neurofilaments were found to be lacking in protein kinase activity when either histone FIIA or the neurofilaments themselves were used as acceptors. There was no augmentation of activity in the presence of cyclic AMP. Addition of microtubule proteins prepared by cycles of assembly and disassembly resulted in phosphorylation of histone, phosphorylation of tubulin and the microtubule-associated proteins, and phosphorylation of neurofilament subunits. The phosphorylation of neurofilaments was predominantly in the 150,000-dalton species and was completely cyclic AMP dependent.

Monoclonal antibodies (mAB) were raised to be used as probes to identify cytoplasmic components associated with intermediate filaments (IF). Four hybridomas (B27, B76, B78, and B100) secreting mAB were generated by fusing mouse myeloma cells with the spleen cells of mice immunized intraperitoneally with Triton-high salt insoluble materials from BHK-21 cells. This insoluble material consists mostly of IF, a small number of microfilaments, and some polyribosomes. Biochemical studies show that the Triton-insoluble materials contain many proteins, including vimentin (decamin) and desmin. Immunofluorescence microscopy of BHK-21 cells stained with the four mAB showed that these mAB decorate the IF in a dotted pattern. Double staining with polyclonal antibody to vimentin confirmed the reactivity of the mAB with the IF. These mAB also stained the vimentin-containing filament system in a variety of other cells including epithelial cells (PTK1 and HeLa) and cells of astroglial origin. Histological studies showed that mAB-B100 stained many types of tissue including epidermis, smooth muscle, and subdermis pericytes, but not the white matter nor the gray matter of the cerebellum and spinal cord. Immunoelectron microscopy with colloidal gold has shown that the mAB-B100 decorated the IF in clusters or aggregates around proteinaceous materials associated with the filaments. Results of immunoprecipitation indicate that mAB-B100 reacted with a protein of 50,000 daltons. These findings suggest that the mAB-B100 we have developed recognizes one of the many components of what appears to be an integrated cytoskeletal structure connected with intermediate filaments.

A soluble isoelectric variant of the 150,000-dalton neurofilament protein was isolated from bovine brain by treating a partially purified filament preparation with a low-ionic-strength high-pH buffer. The protein (S150) had similar peptide maps to the neurofilament component of the same molecular weight (NF150) and was recognized by a polyclonal antibody made against the NF150 polypeptide. However, only half the anti-NF150 activity could be removed with the S150 protein. In addition, the S150 protein had a higher isoelectric point than the NF150 protein. Phosphate analysis indicated that the S150 protein was considerably lessened in phosphate content, which could account for the higher isoelectric point of the protein. It appears, therefore, that the S150 protein may be a precursor of NF150 or the result of phosphatase activity during the isolation procedure. Assembly studies showed that the S150 protein, unlike the NF150 protein, could not assemble with the 70-kDa neurofilament protein, indicating that the phosphate groups which were removed are important in the association of this protein to the neurofilament. When filaments containing all three triplet neurofilament polypeptides or those composed of the 70- and 150-kDa neurofilament proteins were subjected to acid phosphatase, a soluble fraction was obtained, which contained isoelectric variants with higher pI values than the NF150 polypeptide. Only unmodified NF150 protein was found in the insoluble fraction. These results support the argument that removal of phosphate groups results in the dissociation of this protein from the filament.

We have examined the relationship of the ubiquitous 68-70-kDa cytoskeletal-associated protein beta-internexin (Napolitano, E. W., Pachter, J. S., Chin, S. S. M., and Liem, R. K. H. (1985) J. Cell Biol. 101, 1323-1331) to heat-shock cognate 70 (hsc70), the major constitutive member of the mammalian heat-shock protein 70 (hsp70) family of stress proteins. We purify beta-internexin from rat brain microtubules and confirm its identity with hsc70 and the clathrin-uncoating ATPase by the following criteria: 1) The partial sequence of a cyanogen bromide-derived peptide from beta-internexin matches the inferred amino acid sequence of the cDNA clone pRC62 encoding hsc70 from rat brain (O'Malley, K., Mauron, A., Barchas, J. D., and Kedes, L. (1985) Mol. Cell. Biol. 5, 3476-3483). 2) Mixing experiments followed by two-dimensional gel analyses reveal the precise co-migration of beta-internexin, the clathrin-uncoating ATPase, and the in vitro translation product of cDNA clone pHSP-4 encoding rat brain hsc70. 3) beta-Internexin is recognized by a monoclonal antibody reactive against the class of hsp70 proteins. 4) beta-Internexin purified from a microtubule-associated protein-enriched fraction of rat brain by virtue of high affinity binding to ATP-agarose possesses clathrin cage-specific ATPase activity.

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.

Tau is a heat-stable microtubule-associated protein which promotes tubulin polymerization. The assembly promoting region of tau was localized using synthetic peptides modeled after domains found in both human and mouse tau. The design of these synthetic peptides was based on the triple repeat motif found in mouse tau. The first peptide, Tau-(187-204), and the second peptide, Tau-(218-235), are capable of promoting the polymerization of tubulin into microtubules, at concentrations above 100 microM. Two other peptides tested, TauR and Tau-(250-267), were not able to promote the assembly of tubulin over a range of concentrations up to 800 microM. TauR is a random analog of Tau-(187-204). Although TauR is unable to promote polymerization, it can modify Tau-(187-204)-induced tubulin assembly.

The antineurofilament antibodies found in the serum of a chimpanzee with experimental Creutzfeldt-Jakob disease reacted specifically with the 200,000-dalton polypeptide of the purified neurofilament triplet. They also reacted strongly with thoroughly characterized neurofilament swellings of proximal axons of spinal cord motoneurons from beta,beta' iminodipropionitrile (IDPN)-intoxicated rats.

In this article we show a Triton-insoluble, intermediate filament-associated protein of approximately 70 kD to be expressed ubiquitously in diverse mammalian cell types. This protein, assigned the name beta-internexin, exhibits extreme homology in each of the various cell lines as demonstrated by identical limited peptide maps, similar mobilities on two-dimensional gels, and detection in Triton-soluble and -insoluble extracts. beta-Internexin also shares some degree of homology with alpha-internexin, an intermediate filament-associated protein isolated and purified from rat spinal cord, which accounts for the immunologic cross-reactivity displayed by these polypeptides. Light microscopic immunolocalization of beta-internexin with a monoclonal antibody (mAb-IN30) reveals it to be closely associated with the vimentin network in fibroblasts. The antigen is also observed to collapse with the vimentin reticulum during the formation of a juxtanuclear cap induced by colchicine treatment. Ultrastructural localization, using colloidal gold, substantiates the affinity of beta-internexin for cytoplasmic filaments and, in addition, demonstrates its apparent exclusion from the intranuclear filament network. We examine also the resemblance of beta-internexin to a microtubule-associated polypeptide and the constitutively synthesized mammalian heat shock protein (HSP 68/70).

Mammalian neurofilaments are composed of three subunit polypeptides with approximate molecular weights of 200 000, 150 000, and 70 000 (P200, P150, and P70). These subunits were separated by ion-exchange chromatography in the presence of 8 M urea. The P200 polypeptide was differentially eluted on a diethylaminoethyl (DEAE) column. The P70 and P150 polypeptides obtained after the DEAE column were separable on a hydroxylapatite column. Under neurofilament assembly conditions, only the P70 polypeptide was able to reassemble into an intermediate filament in the absence of the other two polypeptides. The P150 and P70 polypeptides copolymerized into an intermediate filament, only if P70 was present. These results suggest that the P70 polypeptide forms the core of the filament and the other two polypeptides are tightly associated accessory proteins.

Indirect immunocytochemical staining with antisera raised against purified glial filament protein and a neurofilament polypeptide was used to study cell interactions between astrocytes and neurons dissociated from embryonic and early postnatal cerebellum. Staining with antibodies raised against purified glial filament protein revealed that greater than 99% of all processes present in cerebellar cultures during the 1st wk in vitro were glial in origin. After 1 wk in culture, unstained processes that were presumably neuronal were observed. Stained astroglial processes formed a dense network that served as a template for cerebellar neurons, identified by indirect immunocytochemical localization of tetanus toxin. More than 90% of neurons from postnatal days 1 or 7 were positioned within one cell diameter of a glial process. In contrast, less than 40% of the neurons dissociated from early embryonic cerebellum were located adjacent to a glial process. Staining with antibodies raised against purified glial filament protein also revealed differences in astroglial morphology that were under developmental regulation. Astroglial cells from embryonic cerebellum were fewer in number and had thick, unbranched processes. Those from postnatal day 1 were more slender, branched, and stellate. Those from postnatal day 7 were highly branched and stellate. Some veil-like astroglial processes were also observed in cells from postnatal animals. These morphological changes were also observed when cells from embryonic day 13 were maintained for a week in vitro. No specific staining of embryonic or postnatal cerebellum cells was observed with antibodies raised against purified neurofilament polypeptides.

The forms, disposition, and cytoskeletal contents of astroglia in immature mouse cerebellum were studied by immunocytochemical staining with antisera against two intermediate filament proteins, vimentin (Vim) (58,000 daltons) and glial filament protein (GF) (51,000 daltons). From embryonic (E) Day 15 to postnatal (P) Day 2, Vim is expressed in cells throughout the cerebellar anlage, including radial glia and Bergmann fibers, cells with amorphous shapes and 2-3 processes, and thick longitudinal elements oriented parallel to axons within axon tracts. GF is not expressed during the first few postnatal days, but by P7, there is a dramatic increase in GF-positive astrocyte-like cells in the putative white matter that are more densely stained and more crowded than at any other age. Between P7 and P14 all astrocytes throughout the cerebellum express both Vim and GF. From P21 on, Vim expression is progressively rarer in all astrocytes except for Bergmann fibers, and GF-positive astrocytes become less numerous. These findings raise two issues: (a) the lineage and relationships of cells expressing Vim and GF; (b) Since GF-positive cells appear as axon ingrowth ceases, axons must grow in a terrain comprised of glial cells that have a different cytoskeletal composition (vimentin), reflecting a less differentiated state, than mature astrocytes or than the GF-rich astrocytes that proliferate after injury in adult CNS.

We have used a human glioma cell line (U-251MG) to study the expression and cytoplasmic organization of vimentin (decamin) and the glial filament protein (GFP). Four clones of the parental U-251 cultures were isolated and found to express GFP from 1-2% to 99% of the cells in the population. Double immunofluorescence microscopy with antibodies to vimentin and GFP has shown that, in all four clonal cell lines, vimentin-containing filaments are expressed in most cells as an organized network and, in GFP-positive cells, GFP and vimentin are associated with the same filament network. Immunoelectron microscopy with specific antibodies labeled with colloidal gold particles of various sizes shows that GFP and vimentin are localized in the same filaments. These findings confirm in vitro studies of the copolymerization of subunits of different biochemical nature into the same intermediate filament and suggest the in vivo probability of the coassembly of GFP and vimentin from a possible soluble pool of monomers.

This report describes 100 consecutive patients treated surgically for diverticulitis of the colon. The main indications for operation were recurrent attacks (33), rapid progressive symptoms (17), bleeding (16), palpable mass (14), or combinations of the above. Eighty-four patients had primary resection (two deaths), ten had staged procedure (two deaths), and six had Hartmann procedures (one death). The mortality was highest in staged procedures as this group of patients included those with complications resulting in the greatest operative risks. No deaths occurred in the elective cases. In 25 cases, various complications developed. The most common was wound infection and sepsis. The five deaths in the series are reported in detail with an evaluation of possible alternative methods of treatment. Follow-up of the series showed only one recurrence in the two years after operation.

Protein hydrogen exchange is generally believed to register some aspects of internal protein dynamics, but the kind of motion at work is not clear. Experiments are being done to identify the determinants of protein hydrogen exchange and to distinguish between local unfolding and accessibility-penetration mechanisms. Results with small molecules, polynucleotides, and proteins demonstrate that solvent accessibility is by no means sufficient for fast exchange. H-exchange slowing is quite generally connected with intramolecular H-bonding, and the exchange process depends pivotally on transient H-bond cleavage. At least in alpha-helical structures, the cooperative aspect of H-bond cleavage must be expressed in local unfolding reactions. Results obtained by use of a difference hydrogen exchange method appear to provide a direct measurement of transient, cooperative, local unfolding reactions in hemoglobin. The reality of these supposed coherent breathing units is being tested by using the difference H-exchange approach to tritium label the units one at a time and then attempting to locate the tritium by fragmenting the protein, separating the fragments, and testing them for label. Early results demonstrate the feasibility of this approach.

Neurofilaments in mammalian nervous tissues have three subunit proteins. These subunit proteins have apparent molecular masses of 200 (NF200), 150 (NF150) and 68 (NF68) kD. Biochemical assembly studies have indicated that the NF68 protein forms the core of the filament and that the other two proteins are associated proteins. Electron microscopy immunolocalization studies have been performed previously on isolated filaments and on filaments from neurons in culture, and have confirmed the localization of NF68 as a core filament protein and NF200 as a peripheral protein. We have raised two monoclonal antibodies to the NF200 components. Using immunogold labelled protein A, we have been able to localize these antibodies to tissue sections of adult cerebellum at the EM level. With this method, we have found that one of the monoclonal antibodies (NF2) shows a linear arrangement of gold particles directly on the filament, whereas the second monoclonal antibody (NF111) reacts with the filaments to give a periodic arrangement of gold particles. By immunoblotting against chymotryptic fragments of the NF200 protein, we have found that the mAB-NF111 reacts solely with a 160 kD piece, whereas the other monoclonal antibody reacts with both the 160 kD piece and the 40 kD piece. The latter piece was shown to be associated to the filament by binding studies with iodinated NF68. Thus the EM localization studies and the biochemical studies indicate that the two monoclonal antibodies react with different parts of the NF200 molecule, one binding to a part of the molecule which is located closer to the filament, and one to a more peripheral part of the molecule.

Two cDNA clones fully encoding the rat neurofilament proteins NF-L and NF-M were subcloned into eukaryotic expression vectors behind the strong constitutive viral promoters from SV40 and Rous sarcoma viruses. Transient transfection of L tk- and Cos cell lines with these expression constructs resulted in cells expressing the neurofilament proteins in an intermediate filament-type pattern. Additionally, a putative juxtanuclear organizing center or region was observed in the transfected cells, most noticeable shortly after the transfection procedure. Stable transfections were performed on mouse L tk- and Swiss 3T6 cells using NF-L and NF-M constructs bearing an SV40 early promoter driven neomycin selectable marker. Although G418-resistant clones were recovered with both the NF-L and the NF-M constructs, only clones expressing immunofluorescently stainable amounts of NF-M were detected and established. Immunoelectron microscopic analysis revealed NF-M and vimentin proteins to be colocalized on the same intermediate filaments.

To study neuron-glial interactions, our laboratory has developed an in vitro model system that, when used with cell type-specific antisera, allows visualization of contacts between cerebellar granule neurons and astroglia. When cells were dissociated from early postnatal mouse cerebellum and plated in microcultures, the neurons aligned along glial filament protein (GFP)-containing astroglial processes. The behavior of the neurons depended on the shape of the particular astroglial cell that they contacted. Neuronal migration commonly occurred along highly elongated astroglial processes of Bergmann-like glia but was inhibited when neurons nestled among the arms of stellate astroglia. To analyze the influence of neurons on the astroglial shapes associated with neuronal migration, cerebellar granule neurons and astroglia were purified and recombined. In the absence of neurons, cerebellar astroglia assumed a flattened shape and proliferated rapidly. In the absence of astroglia, neurite outgrowth was severely impaired. When neurons were recombined with purified astroglia, astroglial proliferation slowed markedly, the shape of the astroglia transformed into complex forms, and neuron-glial interactions were seen. In tissue sections, immature forms of glia were found in the developing cerebellar axon tracts, but no obvious relationship could be discerned between the growing axonal tips and the glia. At P7, a period when the growth of cerebellar axons slows markedly, a transient natural gliosis was seen in the putative white matter. These studies underscore the interdependence of neurons and astroglia during periods of neuron differentiation and neurite outgrowth. In addition, they raise the possibility that the disruption of normal neuronal-astroglial contacts suffered during CNS injury could lead to defects in astroglial form and surface properties that, in turn, might impair axon regrowth.

We describe the complete sequence of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L. Therefore, both the number of introns and their arrangement in the genes coding NF-L and NF-M contrast sharply with the number and arrangement of introns in the genes of known sequence, encoding other members of the intermediate filament multigene family (desmin, vimentin, glial fibrillary acidic protein and the acidic and basic keratins); with the exception of a single truncated keratin gene that lacks an encoded tailpiece, these genes all contain eight introns, of which at least six are placed at homologous locations. Assuming the existence of a primordial intermediate filament gene containing most (if not all) the introns found in contemporary non-neurofilament intermediate filament genes, it seems likely that an RNA-mediated transposition event was involved in the generation of an ancestral gene encoding the NF polypeptides. A combination of insertional transposition and gene-duplication events could then explain the anomalous number and placement of introns within these genes. Consistent with this notion, we show that the genes encoding NF-M and NF-L are linked.

A lambda gtII expression library was prepared from rat brain and screened with a polyclonal antiserum, which recognizes both NF-H and NF-M. An NF-M cDNA clone (pNF-M3C = 1.6 kb) was isolated and characterized. The fusion protein of NF-M3C, when used as an affinity matrix for the anti-neurofilament serum, isolated a subpopulation of antibodies specific for NF-M. Northern analysis demonstrates a single band of approximately 3000 nt and a constant message level for NF-M during postnatal development from postnatal day 0 (PO) to adulthood. Using pNF-M3C as a probe, a second cDNA clone was isolated from a lambda gtII rat brain expression library (pNF-M2D = 2.7 kb). The 2 clones were sequenced and pNF-M2D was found to encode the entire rat NF-M protein. The calculated molecular weight is 95,600, which is only 65% of the molecular weight determined by SDS-PAGE. The amino acid sequence of rat NF-M shows the conserved rod segment present in all intermediate filament proteins. The molecule also contains an unusual C-terminal extension with stretches of glutamic acid, which could contribute to the anomalous migration of this protein on SDS-PAGE and the fact that NF-M does not readily assemble into filaments. The pNF-M2D clone was transcribed and translated in vitro utilizing a rabbit reticulocyte lysate system. The resulting radiolabeled translation products were unexpectedly shown to comigrate with purified rat NF-M on 1- and 2-dimensional gels, even though the translated protein is not phosphorylated.

In order to identify the protein composition of 10 nm neuronal filaments, we prepared enriched fractions of rabbit spinal neurons undergoing experimental neurofilamentous degeneration induced by aluminum. Electron microscopy of the isolated perikarya showed well-preserved, large perinuclear masses of neurofilaments, which were not found in similarly isolated control perikarya. Comparison of these glial-free fractions by SDS-polyacrylamide gel electrophoresis revealed several-fold augmentation in the filament-enriched neurons of proteins migrating at 68,000 and 160,000 daltons, with an additional component at 200,000 daltons. Otherwise, the protein patterns were identical; no band was found at 51,000 daltons, the molecular weight assigned to the major proteins both of glial filaments and of a previously reported bovine brain filament preparation. An antiserum raised against the 160,000 dalton component of a modified bovine brain filament fraction produced specific and intense fluorescent staining of the aluminum-induced neurofilament bundles. Antibodies to the 51,000 dalton protein of brain filaments and to tubulin failed to stain the induced filaments. The results strongly support the hypothesis that both normal and aluminum-induced neuronal filaments are composed of 68,000, 160,000 and 200,000 dalton polypeptides and do not contain significant amounts of the 51,000 dalton filament protein. The likelihood of biochemical heterogeneity among organelles with similar morphology, namely the glial and neuronal filaments, is raised.

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) protein by shifting from a chromaffin-cell-like phenotype to a neurite-bearing sympathetic-neuron-like phenotype. Comparison of the phosphoprotein patterns of the cells by SDS PAGE after various times of NGF treatment revealed a high molecular weight (Mr greater than or approximately 300,000) band whose relative intensity progressively increased beyond 2 d of NGF exposure. This effect was blocked by inhibitors of RNA synthesis and did not require neurite outgrowth or substrate attachment. The enhancement by NGF occurred in serum-free medium and was not produced by exposure to epidermal growth factor, insulin, dibutyryl cAMP, or dexamethasone. Several different types of experiments indicated that this phosphoprotein corresponds to a high molecular weight (HMW) microtubule-associated protein (MAP). These included cross-reactivity with antiserum against brain HMW MAPs, co-cycling with microtubules and co-assembly with tubulin in the presence of taxol. The affected species also co-migrated in SDS PAGE gels with brain MAP1 and, unlike MAP2, precipitated upon boiling. Studies with [35S]-methionine-labeled PC12 cells indicated that at least a significant proportion of this effect of NGF was due to increased levels of protein rather than to mere enhancement of phosphorylation. On the basis of the apparent effects of MAPs on the formation and stabilization of microtubules and of the importance of microtubules in production and maintenance of neurites, it is proposed that induction of a HMW MAP may be one of the steps in the mechanism whereby NGF promotes neurite outgrowth. Furthermore, these findings may lead to an understanding of the role of MAP1 in the nervous system.

The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.

Rosenthal fibers (RFs) are abnormal inclusions within astrocytes, characteristic of Alexander's disease. We have previously isolated a 22 kd protein component of RFs from Alexander's disease brain. By Western blotting, we detected its equivalent in several rat organs, with the highest level in heart, and in a human astrocytoma cell line (U-373MG). A cDNA library established from U-373MG was screened with an anti-RF protein antibody. A partial cDNA clone encoding the lens protein alpha B-crystallin was isolated. The anti-RF protein antibodies react with lens alpha B-crystallin. Furthermore, the distribution of alpha B-crystallin mRNA in rat organs is consistent with the Western blots. Therefore, alpha B-crystallin is not lens-specific and it can accumulate in large amounts in astrocytes in pathological conditions.

In a study of the action of thrombin on fibrinogen and a comparison of this enzyme with trypsin, several fibrinogen-like oligopeptides were synthesized. The hydrolysis of the arginyl-glycine bond in these peptides, by both of these enzymes, is examined and compared.