Biochemical and Biophysical Research Communications, Sep 1, 1990
Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 am... more Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 amino acid residues) containing three cysteines, at positions 7, 86 and 95. Nascent r-hGMF-beta exists in the reduced state and has no biological activity. The protein can be activated through oxidative refolding by incubation with a mixture of reduced and oxidized glutathione. Reverse-phase HPLC analysis of the refolded r-hGMF-beta shows the presence of four peaks, corresponding to the reduced form plus three newly generated intrachain disulfide-containing isoforms predicted from the number of cysteine residues. Only one isoform shows biological activity when tested for growth suppression on C6 glioma cells. We infer from the HPLC elution pattern that the active form contains the disulfide bridge Cys86-Cys95.
Biochemical and Biophysical Research Communications, Sep 1, 1981
Abstract (Na++K+)-ATPase was purified from human kidney of normal and tumor tissue with specific ... more Abstract (Na++K+)-ATPase was purified from human kidney of normal and tumor tissue with specific activities of 100.0 and 16.6 μmol Pi/mg/h, respectively. The antitumor proteins, macromomycin, largomycin, and NSC 327459 (50 μg/ml each) caused 70 to 90% inhibition of (Na++K+)-ATPase from tumor tissue, whereas auromomycin had no effect. (Na++K+)-ATPase from both sources could be phosphorylated by rabbit muscle protein kinase; there was 3 to 6-fold stimulation of phosphorylation by cyclic AMP. Phosphorylation resulted in 70 to 80% decrease in (Na++K+)-ATPase activity, and caused the normal enzyme to become sensitive to inhibition by macromomycin.
Glia maturation factor beta (GMF-beta) is a 17-kDa growth regulating protein isolated from the br... more Glia maturation factor beta (GMF-beta) is a 17-kDa growth regulating protein isolated from the brain. The effect of bovine GMF-beta on neurons was tested on the neuroblastoma line N18 and the pheochromocytoma line PC12. GMF-beta inhibited the proliferation of N18 cells and promoted their neurite outgrowth, with an increase in neurofilament protein, but had no effect on PC12 cells. This was in contrast to nerve growth factor (NGF) which regulated PC12 but not N18. Acidic fibroblast growth factor (FGF), on the other hand, had a weak effect on PC12 but none on N18. Antisera against GMF-beta and NGF neutralized the biological activity of the corresponding growth factors but showed no cross-neutralization. Fluorescence visualization revealed the binding of GMF-beta to N18 cells but not to PC12 cells; the opposite was true with NGF.
Advances in Experimental Medicine and Biology, 1991
ABSTRACT In 1972 this laboratory observed the ability of brain extracts to promote the phenotypic... more ABSTRACT In 1972 this laboratory observed the ability of brain extracts to promote the phenotypic expression of cultured astrocytes and named the active agent “glia maturation factor” (GMF) (Lim et al., 1972, 1973; Lim and Mitsunobu, 1974). Attempts at purifying this factor encountered a number of difficulties. First, it exists in low abundance. Second, the purer the protein is the more unstable it becomes. Third, as purification steps increase, the procedure becomes more and more laborious to carry through. These problems are common to many growth factors and therefore not unique to GMF. However, the purification of GMF was further complicated by the fact that the brain is a source of several other potent growth factors, some of which interfered with the monitoring of GMF.
Biochemical and Biophysical Research Communications, Jun 1, 2002
We infected a mixed culture of primary rat astrocytes and microglia with a replication-defective ... more We infected a mixed culture of primary rat astrocytes and microglia with a replication-defective adenovirus carrying the rat glia maturation factor (GMF) cDNA. Affymetrix microarray analysis showed a big increase in the expression of several major histocompatibility complex (MHC) class II proteins along with interleukin-1beta (IL-1beta). Subsequent study using reverse transcription-polymerase chain reaction (RT-PCR) yielded the same results with the mixed culture, but not with pure astrocytes or pure microglia. We also noticed that the GMF/virus construct infected only astrocytes but not microglia. This led us to suspect that overexpression of GMF in astrocytes resulted in the secretion of an active substance that stimulated the microglia to express MHC II and IL-1beta. We identified this substance as granulocyte-macrophage-colony stimulating factor (GM-CSF). MHC II are unique to antigen-presenting cells such as microglia and monocytes. The results suggest that GMF in astrocytes can initiate a series of events, leading to immune activation in the nervous system, and implicates its involvement in autoimmune diseases such as multiple sclerosis.
Biochemical and Biophysical Research Communications, Sep 1, 1990
Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 am... more Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 amino acid residues) containing three cysteines, at positions 7, 86 and 95. Nascent r-hGMF-beta exists in the reduced state and has no biological activity. The protein can be activated through oxidative refolding by incubation with a mixture of reduced and oxidized glutathione. Reverse-phase HPLC analysis of the refolded r-hGMF-beta shows the presence of four peaks, corresponding to the reduced form plus three newly generated intrachain disulfide-containing isoforms predicted from the number of cysteine residues. Only one isoform shows biological activity when tested for growth suppression on C6 glioma cells. We infer from the HPLC elution pattern that the active form contains the disulfide bridge Cys86-Cys95.
Biochemical and Biophysical Research Communications, Sep 1, 1981
Abstract (Na++K+)-ATPase was purified from human kidney of normal and tumor tissue with specific ... more Abstract (Na++K+)-ATPase was purified from human kidney of normal and tumor tissue with specific activities of 100.0 and 16.6 μmol Pi/mg/h, respectively. The antitumor proteins, macromomycin, largomycin, and NSC 327459 (50 μg/ml each) caused 70 to 90% inhibition of (Na++K+)-ATPase from tumor tissue, whereas auromomycin had no effect. (Na++K+)-ATPase from both sources could be phosphorylated by rabbit muscle protein kinase; there was 3 to 6-fold stimulation of phosphorylation by cyclic AMP. Phosphorylation resulted in 70 to 80% decrease in (Na++K+)-ATPase activity, and caused the normal enzyme to become sensitive to inhibition by macromomycin.
Glia maturation factor beta (GMF-beta) is a 17-kDa growth regulating protein isolated from the br... more Glia maturation factor beta (GMF-beta) is a 17-kDa growth regulating protein isolated from the brain. The effect of bovine GMF-beta on neurons was tested on the neuroblastoma line N18 and the pheochromocytoma line PC12. GMF-beta inhibited the proliferation of N18 cells and promoted their neurite outgrowth, with an increase in neurofilament protein, but had no effect on PC12 cells. This was in contrast to nerve growth factor (NGF) which regulated PC12 but not N18. Acidic fibroblast growth factor (FGF), on the other hand, had a weak effect on PC12 but none on N18. Antisera against GMF-beta and NGF neutralized the biological activity of the corresponding growth factors but showed no cross-neutralization. Fluorescence visualization revealed the binding of GMF-beta to N18 cells but not to PC12 cells; the opposite was true with NGF.
Advances in Experimental Medicine and Biology, 1991
ABSTRACT In 1972 this laboratory observed the ability of brain extracts to promote the phenotypic... more ABSTRACT In 1972 this laboratory observed the ability of brain extracts to promote the phenotypic expression of cultured astrocytes and named the active agent “glia maturation factor” (GMF) (Lim et al., 1972, 1973; Lim and Mitsunobu, 1974). Attempts at purifying this factor encountered a number of difficulties. First, it exists in low abundance. Second, the purer the protein is the more unstable it becomes. Third, as purification steps increase, the procedure becomes more and more laborious to carry through. These problems are common to many growth factors and therefore not unique to GMF. However, the purification of GMF was further complicated by the fact that the brain is a source of several other potent growth factors, some of which interfered with the monitoring of GMF.
Biochemical and Biophysical Research Communications, Jun 1, 2002
We infected a mixed culture of primary rat astrocytes and microglia with a replication-defective ... more We infected a mixed culture of primary rat astrocytes and microglia with a replication-defective adenovirus carrying the rat glia maturation factor (GMF) cDNA. Affymetrix microarray analysis showed a big increase in the expression of several major histocompatibility complex (MHC) class II proteins along with interleukin-1beta (IL-1beta). Subsequent study using reverse transcription-polymerase chain reaction (RT-PCR) yielded the same results with the mixed culture, but not with pure astrocytes or pure microglia. We also noticed that the GMF/virus construct infected only astrocytes but not microglia. This led us to suspect that overexpression of GMF in astrocytes resulted in the secretion of an active substance that stimulated the microglia to express MHC II and IL-1beta. We identified this substance as granulocyte-macrophage-colony stimulating factor (GM-CSF). MHC II are unique to antigen-presenting cells such as microglia and monocytes. The results suggest that GMF in astrocytes can initiate a series of events, leading to immune activation in the nervous system, and implicates its involvement in autoimmune diseases such as multiple sclerosis.
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