Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons.

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.

Neurotrophic protein regulates muscle acetylcholinesterase in culture.

Skeletal muscles lose acetylcholinesterase in culture as a result of denervation. A protein fraction isolated from peripheral nerves maintained the level of acetylcholinesterase in cultures of aneural embryonic muscle or denervated adult chicken muscle. These results indicate that trophic regulation of muscle acetylcholinesterase might be mediated by a protein produced by nerves.

Transferrin: assay of myotrophic effects and method for immunocytochemical localization.

1. Primary cultures of dissociated embryonic chicken skeletal muscle cells provide an ideal model for investigating the effects of growth factors such as Tf because these cells undergo a highly integrated pattern of differentiation and maturation. 2. The trophic effects of a growth factor such as Tf can be assessed on muscle cultures by the determination of such parameters as acetylcholinesterase and acetylcholine receptors. These proteins are specific to the cultured myotubes, appear in high levels following fusion of myoblasts into myotubes, and are relatively easy to assay. 3. Tf and other growth factors are internalized by a receptor-mediated mechanism (see Trowbridge et al. and Seligman and Allen, this volume). These growth factors can be localized to specific tissues by immunocytochemistry at the light or electron microscopic level. This information on cellular distribution could be very useful in assessing the pattern of growth and differentiation with regard to the particular growth factor under study.

Immunocytochemical localization of mitochondrial malate dehydrogenase in primary cultures of rat astrocytes and oligodendrocytes.

To assess the oxidative metabolism of glial cells, we visualized mitochondrial malate dehydrogenase (mMDH) in purified cultures of neonatal rat polygonal and process-bearing astrocytes as well as in oligodendrocytes, using indirect immunofluorescence. Double immunofluorescent localization of rabbit anti-mMDH and either mouse monoclonal antiglial fibrillary acidic protein or anti-myelin basic protein demonstrated that both process-bearing astrocytes and oligodendrocytes showed uniformly intense anti-mMDH immunoreactivity in their cell bodies. However, immunoreactivity to mMDH among polygonal astrocytes varied from very weakly positive to intensely positive. Experiments with rhodamine 123, a mitochondrion-specific fluorochrome, indicated that polygonal astrocytes contain relatively similar numbers of mitochondria; this suggested that the variable intensities of anti-mMDH immunoreactivity observed did not result from differences in mitochondrial numbers. In cultures of polygonal astrocytes maintained in a chemically defined medium containing growth factors and hormones, or in complete culture medium containing 1mM N6, O2-dibutyryl adenosine 3',5'-cyclic phosphate, the resultant stellate astrocytes still showed their original variable levels of anti-mMDH immunoreactivity. This suggested that the mMDH distribution pattern did not depend on the degree of morphological differentiation. Furthermore, cultures of polygonal astrocytes isolated from four specific regions of neonatal rat brain showed variable but reproducible profiles of anti-mMDH immunoreactivity. Our results suggest that there may be an appreciable range in the level of oxidative metabolism among individual polygonal astrocytes in culture.

Immunocytochemical localization of aldolase in normal, denervated, and dystrophic chicken muscles.

To investigate whether immunocytochemical localization of muscle-specific aldolase can be used for fiber phenotype determination, we produced specific antibodies against the enzyme and studied its distribution in adult chicken skeletal muscles by indirect immunofluorescence microscopy. Monoclonal antibodies against the myosin heavy chains of fast-twitch (MF-14) and slow-tonic (ALD-58) muscle fibers were also used to correlate aldolase levels with the fiber phenotype. The goat anti-aldolase antibody was found to be specific for the A form of aldolase, as evidenced by sodium dodecyl sulfate gel electrophoresis, immunotitration experiments, and immunoblot analysis. The antibody reacted strongly with the fast-twitch myofibers of normal pectoralis and posterior latissimus dorsi muscles; the phenotype of these muscle fibers was confirmed by a positive immunofluorescent reaction after incubation with MF-14 antibody. By contrast, the slow-tonic myofibers of normal anterior latissimus dorsi, which react positively with ALD-58 antibody, reacted weakly with anti-aldolase antibodies. In denervated chicken muscles, reaction to anti-aldolase antibodies was markedly reduced in fast-twitch fibers, although reaction to MF-14 was not diminished. By contrast, in dystrophic muscle, fast-twitch fibers showed reduced reactivity to anti-aldolase and marked to moderate reduction in MF-14 reactivity. Our results show that: (a) in normal muscles, reactivity to anti-aldolase matches the phenotype obtained by using anti-fast or anti-slow myosin heavy chain antibodies, and therefore can serve to identify mature fibers as fast or slow; and (b) in denervated or dystrophic muscles, the intracellular expressions of aldolase and fast-twitch myosin heavy chains are regulated independently.

Sciatin: immunocytochemical localization of a myotrophic protein in chicken neural tissues.

A mytotrophic protein (sciatin) purified from chicken sciatic nerves has "trophic" or "maintenance" effects on cultured muscle. We have elicited a specific antiserum against purified sciatin in rabbits. Using this antiserum, we investigated the distribution of sciatin in embryonic and adult chicken tissues by an unlabeled peroxidase-an-tiperoxidase method at the light microscopic level. The antiserum stained adult chicken neural tissues in situ and cultured embryonic chick neurons. Staining was intense in the cell bodies of spinal cord neurons and the axoplasm of sciatic nerves. These was reaction product seen in the outer margins of myelin sheaths that corresponded to the Schwann cell cytoplasm. Cerebral cortical neurons were weakly stained by the antiserum. No staining was apparent in oligodendrocytes or astrocytes. Nonneural tissues, such as skeletal, smooth and cardiac muscle, kidney, and liver, were also unstained by the antiserum. Cultured spinal cord neurons, cerebral cortical neurons, and sensory neurons were stained immunocytochemically by the antiserum. There was no reaction product seen in the glial cells that are usually present in neuronal cultures or cultured cells from liver, kidney, skeletal muscle, smooth muscle, and cardiac muscle. Our results thus show that the myotrophic protein is localized in neuronal perikarya and their processes in vivo as well as in vitro.

Neural control of muscle.

Cholinergic innervation regulates the physiological and biochemical properties of skeletal muscle. The mechanisms that appear to be involved in this regulation include soluble, neurally-derived polypeptides, transmitter-evoked muscle activity and the neurotransmitter, acetylcholine, itself. Despite extensive research, the interacting neural mechanisms that control such macromolecules as acetylcholinesterase, the acetylcholine receptor and glucose 6-phosphate dehydrogenase remain unclear. It may be that more simplified in vitro model systems coupled with recent dramatic advances in the molecular biology of neurally-regulated proteins will begin to allow researchers to unravel the mechanisms controlling the expression and maintenance of these macromolecules.

Rapid increase in immunoreactivity to GFAP in astrocytes in vitro induced by acidic pH is mediated by calcium influx and calpain I.

In higher vertebrates, reactive gliosis resulting from injury to the central nervous system (CNS) is characterized by a rapid increase in immunoreactivity (IR) to glial fibrillary acidic protein (GFAP). Little is known about the extracellular signals that initiate the increase in GFAP-IR following CNS injury. We demonstrated recently [T.H. Oh, G.J. Markelonis, J.R. Von Visger, B. Baik, M.T. Shipley, Acidic pH rapidly increases immunoreactivity of glial fibrillary acidic protein in cultured astrocytes, Glia 13 (1995) 319-322] that a rapid increase in GFAP-IR can be evoked in mature astrocyte cultures by exposing the cells to an acidic medium. We investigated the intracellular pathway(s) involved in initiating increased GFAP-IR, a hallmark of reactive astrocytes. The increase in GFAP-IR produced by exposure to acidic medium was blocked by pretreatment with nickel ions, by such blockers of L-type calcium channels as nifedipine, verapamil and diltiazem, by calpain inhibitor I, or by the intracellular calcium chelator, BAPTA-AM. At physiological pH, treatment with the calcium ionophore, A23187, resulted in increased GFAP-IR which could be blocked by pretreatment with calpain inhibitor I. Astrocytes exposed to low pH exhibited a marked increase in a GFAP fragment with a molecular weight of 48 kDa. In astrocytes exposed to acidic medium, alpha-fodrin, a selective endogenous substrate of calpain, was also found to be hydrolyzed producing fragments with molecular weights of 120-150 kDa. As anticipated, pretreatment with calpain inhibitor I prevented the proteolytic degradation of both GFAP and alpha-fodrin in these samples. These results suggest that the initial increase in GFAP-IR after CNS injury appears to be linked to Ca(++) influx, and is mediated further by a proteolytic process that seemingly involves the activation of the calcium-dependent protease, calpain I.

Immunocytochemical distribution of transferrin and its receptor in the developing chicken nervous system.

Transferrin is the plasma protein responsible for iron transport in all vertebrates. While transferrin is known to have growth-promoting activity on a variety of cells in culture, the role of transferrin and its membrane receptor in neuronal development is unknown. Using antibodies to transferrin and transferrin receptors, we studied the immunocytochemical localization of transferrin and its receptor in developing chicken neural tissues by the peroxidase-antiperoxidase method. In 5-day-old embryonic brain, germinal cells of the ventricular zone showed a positive reaction for transferrin receptors but were negative for transferrin. By 6-7 days, transferrin-positive cells were seen in the inner layer of the ventricular zone and a few 'patches' of transferrin-positive cells were also seen in the adjacent area. By 10 days, large neurons throughout the brain were strongly positive for transferrin. By 11-16 days, all neurons in the brain showed a strong positive reaction for the protein. Thereafter, the transferrin-positive reaction became gradually weaker in neurons whereas the walls of blood capillaries showed a positive reaction for transferrin. In the adult brain, neurons showed very weak or negative staining. A similar staining pattern for transferrin was observed in the developing spinal cord and dorsal root ganglia (DRG). By 10-12 days, both spinal cord neurons and DRG neurons showed strong reactions for transferrin. Thereafter, the transferrin-positive reaction gradually diminished in older spinal cord neurons and completely disappeared from DRG neurons. Cultured cerebral hemisphere, spinal cord, and DRG neurons showed positive staining reactions for both transferrin and its receptor. Our results suggest that: transferrin is initially taken up by developing neurons from cerebrospinal fluid via receptor-mediated endocytosis; the accumulation of transferrin ultimately reaches a maximum level within immunoreactive neurons and then declines just prior to hatching; in contrast to other CNS neurons, DRG neurons accumulate transferrin only briefly and then become negative for transferrin by immunocytochemistry; and after closure of the blood-brain barrier, transferrin may reach neurons by transport across capillaries into the 'paravascular' spaces. In view of these results, transferrin may play some important but unrecognized role in early neuronal development in vivo as well as in vitro.

Receptor-mediated uptake of labeled transferrin by embryonic chicken dorsal root ganglion neurons in culture.

Transferrin is a growth-promoting plasma protein which is known to occur within developing neurons. Since little information exists on the process by which transferrin is internalized by neurons, we studied this process using dissociated embryonic chicken dorsal root ganglion neurons in culture. Cultured dorsal root ganglion neurons were incubated in the presence of 3.75 nM (125)I-transferrin at 37°C, the cultures were extensively washed, the neurons were solubilized in a Triton-containing buffer and internalized (125)I-transferrin was quantified with a gamma counter. (125)I-transferrin was internalized in a linear fashion for at least 60 min, and this uptake was abolished by the presence of 1.25 µM unlabeled transferrin. No competition for the uptake of (125)I-transferrin was observed in the presence of 1.25 µM ovalbumin, cytochrome c, hemoglobin, insulin, horseradish peroxidase, aldolase or the carboxyl-terminal fragment ('half-site') of transferrin. By contrast, uptake was inhibited by approximately 50% in the presence of the ammo-terminal fragment ('half-site') of transferrin (1.25 µM) or in the presence of concanavalin A (1.25 µM). The binding of transferrin conjugated to fluorescein isothiocyanate to neurons at 4°C and its subsequent internalization at 37°C was demonstrated by fluorescence microscopy of unfixed cells following incubation of the neurons in the presence of the fluorescently labeled protein. Furthermore, the transferrin receptors were visualized immunocytochemically on the surface membranes of dorsal root ganglion neurons using rabbit antibodies directed against transferrin receptors from chicken reticulocytes. From these data, we conclude that transferrin is internalized by neurons via receptor-mediated endocytosis, and suggest that this protein may serve an important role in the development and survival of dorsal root ganglion neurons.

Neuroglial responses to the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mouse striatum.

We have studied the reactive responses of both astrocytes and microglia to dopaminergic denervation of the striatum by MPTP. Following MPTP treatment, increased GFAP immunoreactivity reached a peak at 2 days and persisted for at least 6 weeks. Immunoreactivity to vimentin was also markedly increased in astrocytes 48 h after MPTP treatment. Striatal laminin immunoreactivity, however, appeared to be unaffected by drug treatment. GFAP protein levels increased to 196% and 321% of control 24 and 48 hours after MPTP treatment, respectively. Concomitantly, GFAP mRNA levels increased to 560% and 1620% of control, respectively. These reactive changes in striatal astrocytes in response to MPTP treatment were also accompanied by a reactive microglial response as evidenced by increased immunohistochemical visualization of striatal microglia using antibodies to Mac-1. Our results and those reported previously by O'Callaghan et al., strongly suggest that MPTP-induced reactive gliosis in mouse striatum is associated with reactive microglia, albeit without increased interleukin-1 beta.

Localization of transferrin within the developing vertebrate nervous system.

Transferrin is one of several serum proteins localized within neurons during development of the nervous system. The expression of transferrin receptors appears to precede the active accumulation of transferrin by neurons. The first cells immunoreactive for transferrin appear adjacent to the ventricles or to the central canal of the spinal cord. These cells then appear to migrate from this site. These neurons become progressively more immunoreactive for transferrin, attain a peak of reactivity and then lose their reaction to antitransferrin antibodies. Thus, a "window" of transferrin immunoreactivity is found. As neurons lose their reactivity to antitransferrin antibodies, glia and the walls of capillaries become positive. In the rat nervous system, the gradual decrease in intraneuronal transferrin is accompanied by an increase in mitochondrial malate dehydrogenase, an enzyme of the tricarboxylic acid cycle. Thus, the accumulation of transferrin appears to closely precede the ontogeny of oxidative metabolism in the brain. As transferrin appears transiently in all neurons, this protein may be involved in a number of other important developmental events such as the expression of dopamine D2 receptors and the period of "programmed" cell death in the spinal cord.

Chicken serum transferrin duplicates the myotrophic effects of sciatin on cultured muscle cells.

Sciatin, a glycoprotein purified from chicken sciatic nerves, has been shown to have trophic effects on chicken skeletal muscle cells in culture. Since we recently observed pronounced structural similarities between sciatin and chicken serum transferrin [Markelonis et al, 1982a], we decided to investigate the muscle growth-promoting activity of transferrin on cultured muscle cells. Serum transferrin was isolated by the same protocol used to purify sciatin, viz., affinity chromatography on concanavalin A-agarose followed by ion-exchange chromatography on DEAE cellulose. The serum protein recovered by this purification scheme was indistinguishable immunologically from sciatin as evidenced by a positive precipitin reaction against goat anti-sciatin serum on double immunodiffusion in agar. Purified serum transferrin had myotrophic effects identical to those of sciatin when added to skeletal muscle cells in vitro. For example, even when chicken embryo extract--a constituent normally required for chicken muscle cell differentiation in vitro--was omitted from culture medium, either serum transferrin or sciatin promoted myogenesis in culture as measured by a stimulation of the fusion index. Furthermore, both proteins caused a significant increase in the level of protein synthesis, the number of acetylcholine receptors and the activity of acetylcholinesterase in treated muscle cultures. By contrast, commercially obtained ovotransferrin (conalbumin) or FeSO4 (100 microM) were unable to fully support myogenesis of skeletal muscle in vitro if embryo extract was omitted from the culture medium. From these data, we conclude that the neuronal myotrophic protein sciatin is both structurally and biologically related to serum transferrin. Furthermore, we suggest that sciatin may represent a neuronal form of this iron-transport protein.

Dependence of in vitro myogenesis on a trophic protein present in chicken embryo extract.

A trophic protein (sciatin) purified from sciatic nerves has been shown to enhance the morphological development and to promote the maintenance of skeletal muscle cells in vitro [Markelonis, G. J. & Oh, T. H. (1979) Proc. Natl. Acad. Sci. USA 76, 2470-2474]. We have elicited a specific antiserum against purified sciatin in rabbits. By using this antiserum, we have examined whether sciatin is also required for the initial differentiation of avian myogenic cells in vitro. Sciatin was found to be a component of chicken embryo extract, a constituent of culture medium required for myogenesis in vitro, by an immunodiffusion assay and by NaDodSO4 gel electrophoresis of immunoprecipitates. The removal of sciatin from chicken embryo extracts by immunoprecipitation with antiserum against sciatin completely inhibited myogenesis. When myogenic cells were grown in culture medium from which sciatin had been removed, the cells failed to differentiate beyond the myoblast stage. However, when sciatin (25 microgram/ml) was added to the sciatin-absorbed culture medium, normal myogenesis ensued. Furthermore, myogenic cells underwent normal myogenesis in the absence of embryo extract if sciatin (25 microgram/ml) was added to the culture medium. These results demonstrate that sciatin is the component of chicken embryo extract required for myogenesis and that the protein influences the initial differentiation of myogenic cells in vitro.

Purification of sciatin using affinity chromatography on concanavalin A-Agarose.

A glycoprotein from chicken sciatic nerves, sciatin, has been shown to have trophic effects on the maturation and maintenance of skeletal muscle cells in culture. This protein was purified 24-fold from sciatic nerve extracts by affinity chromatography on concanavalin A-agarose followed by ion-exchange on diethylaminoethyl cellulose. The purity of sciatin obtained by this procedure was greater than 97% as estimated by densitometric integration of sodium dodecyl sulfate gels, and represented 33% of the sciatin present in sciatic nerve extracts as determined by rocket immunoelectrophoresis. Sciatin purified by this technique retained full biological activity since (1) addition of the protein to embryonic chicken skeletal muscle cells in culture enhanced the morphological development of the cells, and (2) the protein increased the number of acetylcholine receptors as measured by binding of 125I-alpha-bungarotoxin to 261% of the control value after 4 days in vitro. The purification procedure described in the present communication provides a more rapid and convenient method for the isolation of this trophic protein.

Effects of interleukin-1 beta and tumor necrosis factor-alpha on the expression of glial fibrillary acidic protein and transferrin in cultured astrocytes.

Recent evidence suggests that interleukin (IL)-1 and tumor necrosis factor (TNF) may play a role in astrogliosis following injury to the CNS. The short-term biochemical effects of these immune-related cytokines were determined on cultured rat polygonal and process-bearing astrocytes. Both IL-1 and TNF stimulated the rate of thymidine incorporation in polygonal astrocytes up to 137% and 215%, respectively, over the level observed in untreated controls. By contrast, thymidine incorporation was relatively unaffected by these cytokines in process-bearing astrocytes. The cytokines did not significantly affect the level of glial fibrillary acidic protein (GFAP) within polygonal astrocytes, even though they appeared to downregulate the expression of GFAP mRNA by as much as 62%. Both cytokines increased the intracellular expression of transferrin (Tf) within some polygonal astrocytes. In untreated control cultures, fewer than than 2% of polygonal astrocytes were immunoreactive for Tf. By contrast, approximately 30% of polygonal astrocytes treated with IL-1 or TNF-alpha became strongly immunoreactive for Tf. Neither IL-2 nor a number of other known growth factors appeared to alter the level of immunoreactive Tf in these cells. Process-bearing astrocytes were negative for Tf, regardless of the treatment used. Northern blot analysis demonstrated that the level of Tf mRNA in cultures of polygonal astrocytes increased 148% above the level observed in untreated controls following treatment with either IL-1 or TNF, whereas no change was observed following treatment with IL-2. These results suggest that increased levels of particular cytokines known to be present in injured CNS can produce pronounced biochemical alterations within a subtype of cultured astrocytes.

A neurite-promoting factor from muscle supports the survival of cultured chicken spinal motor neurons.

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.

Sciatin: purification and characterization of a myotrophic protein from chicken sciatic nerves.

A protein isolated from sciatic nerves of adult chickens promotes the morphological maturation and maintenance of embryonic avian skeletal muscle cells in the absence of innervation and is required for normal myogenesis in vitro. This trophic protein, sciatin, has been purified by ion exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-100. Sciatin migrated as a single polypeptide chain of molecular weight 84,000 on sodium dodecyl sulfategel electrophoresis. The native molecular weight of sciatin as determined by sedimentation equilibrium centrifugation was 86,400. Amino acid analysis revealed that sciatin is relatively deficient in tryptophan, histidine, glycine, and arginine, but enriched in cysteine, methionine, alanine, and lysine. Carbohydrate determination showed that sciatin in composed of 11% sugar by weight with no detectable N-acetylneuraminic acid residues. Sedimentation velocity centrifugation studies revealed an S20,w0 of 5.11 with a frictional coefficient of 1.31. Sciatin had no detectable protease or acetylcholinesterase activity. The results of the present study provide new biochemical information on a macromolecule with biological activities similar to those expressed by the "maintenance" group of growth factors which includes such proteins as nerve growth factor.

Effects of cell division, cell density, and cyclic nucleotides on choline acetyltransferase activity in a cholinergic neuroblastoma cell line (S-20Y).

We investigated the effects of a number of experimental perturbations on choline acetyltransferase (ChAT) in a cholinergic mouse neuroblastoma cell line (S-20Y). ChAT specific activity increased by 4.5-fold during growth, suggesting that enzyme activity is dependent on increased cell density. This was confirmed by assessing enzyme activity at differential initial seeding densities. ChAT activity was also markedly enhanced by 1 mM dibutyryl cyclic-3',5'-AMP (dBcAMP), an effect that was blocked by cycloheximide. Confirmation of the dBcAMP effect was achieved with forskolin, a compound known to enhance intracellular cyclic AMP; forskolin (100 microM) caused a significant increase in ChAT activity. After a 20-h latent interval ChAT activity was also enhanced significantly by cytosine arabinoside. The common element in these diverse effects on ChAT activity may be cessation of cell division, although cell-cell interactions at the level of the cell membrane may also be important in the control of ChAT in S-20Y.