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G J Markelonis

Publications and source records attributed to G J Markelonis.

35 records · Page 2Linked to original sources
PubMed1988

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.

Animals↗
PubMed1987

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.

Acetylcholinesterase↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1985

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.

Animals↗
PubMed1982

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.

Animals↗
PubMed1982

Sciatin is a transferrin-like polypeptide.

Sciatin, an acidic glycoprotein from chicken sciatic nerve, the myotrophic effects on avian skeletal muscle cells in culture. As sciatin was found to have certain structural similarities to transferrin, we further investigated the physiochemical characteristics of sciatin in order to determine the relationship between these two proteins. Sciatin was found to be strikingly similar to ovotransferrin in amino acid composition. In addition, amino acid sequence analysis revealed that sciatin and ovotransferrin and identical amino-terminal sequences for a t least the first 20 amino acid residues. Chicken ovotransferrin, but not human serum transferrin, cross-reacted with rabbit antisciatin antibodies upon rocket immunoelectrophoresis and double immunodiffusion in agar. In addition, in the presence of bicarbonate, sciatin bound approximately 2 mol ferrous iron/mol protein. Using the purification procedure developed for sciatin, we purified a protein from chicken serum that cross-reacted with antisciatin serum, migrated at a position identical to that of sciatin or ovotransferrin on two-dimensional gel electrophoresis, had an amino composition very similar to ovotransferrin and sciatin, and had myotropic effects on cultured muscle cells. From these data, we conclude that sciatin is a growth-promoting polypeptide closely related in structure to transferrin.

Amino Acid Sequence↗
PubMed1981

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.

Animals↗
PubMed1981

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.

Animals↗
PubMed1980

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.

Acetylcholinesterase↗
PubMed1980

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.

Animals↗
PubMed1978

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.

Acetylcholinesterase↗
PubMedDate not supplied

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.

Animals↗