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R K Liem

Publications and source records attributed to R K Liem.

At least 91 records · Page 5Linked to original sources
PubMed1984

Identification of glial filament protein and vimentin in the same intermediate filament system in human glioma cells.

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.

Astrocytoma↗
PubMed1984

Two forms of cerebellar glial cells interact differently with neurons in vitro.

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.

Animals↗
PubMed1984

Neuron-astroglial interactions in vitro and their implications for repair of CNS injury.

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.

Animals↗
PubMed1984

Characterization of antineurofilament autoantibodies in Creutzfeldt-Jakob disease.

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.

Animals↗
PubMed1984

Promotion of microtubule assembly by neurofilament-associated microtubule-associated proteins.

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.

Animals↗
PubMed1984

Defects in specific associations between astroglia and neurons occur in microcultures of weaver mouse cerebellar cells.

The neurological mutation weaver is characterized by defects in granule cell migration along Bergmann glial processes and by subsequent death and disposition of granule cells. Immunocytochemical localization of antisera raised against purified glial filament protein (AbGF) and transmission electron microscopy were used to visualize specific associations between granule neurons and astroglia in microcultures of cerebellar cells dissociated from normal (+/+), heterozygous (+/wv), and homozygous (wv/wv) B6CBA-w mouse cerebella. In microcultures of cells dissociated from normal B6CBA-Aw-J-wv (+/+) cerebella, staining with AbGF closely resembled results previously reported for cells taken from C57BL/6J (+/+) tissue. Two forms of stained astroglia were seen, one with a larger perikaryon and shorter processes, among which 12 to 20 unstained cells nestled; and another with a smaller cell soma and longer processes, along which a few unstained cells were seen. The first resembled astrocytes of the internal granular layer and white matter, the second Bergmann astroglia. In microcultures of heterozygous animals (+/wv), the number of granule cells was reduced slightly. Many stained astroglia resembled those from +/+ cerebella, but others had thickened processes and enlarged terminal "endfeet." Granule cells associated with both forms of stained astroglia. Cultures from homozygous weaver (wv/wv) cerebella contained very few, if any, granule cells and did not exhibit specific neuronal/glial interactions characteristic of +/+ cells. Both forms of stained wv/wv glial cells had enlarged cell somata giving rise to stunted processes, suggesting that both Bergmann glia and astrocytes are affected by the weaver gene. By both immunocytochemical staining and electron microscopy, accumulation and tangling of glial filaments were seen. Immunocytochemical staining of weaver cerebellar tissue with AbGF also revealed abnormalities in astrocytes of the internal granular layer and white matter in addition to previously reported defects in Bergmann glia. Agglutination with five plant lectins, each with a different carbohydrate-binding specificity, revealed that postnatal weaver cerebellar cells agglutinate with the lectins concanavalin A and the wheat germ agglutinin, suggesting the persistence postnatally of embryonic cell surface elements on postnatal weaver cells.

Animals↗
PubMed1983

A batchwise purification procedure of neurofilament proteins.

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.

Animals↗
PubMed1983

Regulation of a high molecular weight microtubule-associated protein in PC12 cells by nerve growth factor.

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.

Animals↗
PubMed1983

An intermediate filament-associated protein, p50, recognized by monoclonal antibodies.

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.

Animals↗
PubMed1982

Purification of individual components of the neurofilament triplet: filament assembly from the 70 000-dalton subunit.

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.

Animals↗
PubMed1982

Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging.

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.

Animals↗
PubMed1981

Astroglial cells provide a template for the positioning of developing cerebellar neurons in vitro.

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.

Animals↗
PubMed1981

Preferential phosphorylation of the 150,000 molecular weight component of neurofilaments by a cyclic AMP-dependent, microtubule-associated protein kinase.

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.

Animals↗
PubMed1980

A high energy structure change in hemoglobin studied by difference hydrogen exchange.

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.

Acetamides↗
PubMed1980

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

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.

Deuterium↗