Ramon Lim

Maturation-stimulating effect of brain extract and dibutyryl cyclic AMP on dissociated embryonic brain cells in culture

Abstract Extracts from adult rat brains contain a macromolecular factor capable of transforming dissociated embryonic rat brain cells in a monolayer culture. The factor simulates the activity of dibutyryl cyclic AMP but is not identical with either cyclic AMP or prostaglandin.

Rat astrocytes and Schwann cells in culture synthesize nerve growth factor-like neurite-promoting factors.

Neurite-promoting activity in feeding medium conditioned by rat astrocytes and Schwann cells in culture was examined. The conditioned medium (CM) from both types of glial cultures stimulated extensive neurite outgrowth from embryonic chick dorsal root ganglia (DRG) as well as pheochromocytoma (PC12) cells. Both the DRG and PC12 cells also produce neurite outgrowth in the presence of nerve growth factor (NGF). With the DRG, the neurite growth rates observed with the glial cell CM were identical to growth rates seen with NGF. Although anti-NGF antibody did not inhibit the neurite outgrowth produced by either of the glial CM, a nerve growth factor radioreceptor assay did detect an NGF-like molecule in both CM. Since the extensive neurite outgrowth stimulated by the glial CM was not mimicked by pure laminin alone, we conclude that the glial neurite promoting factors are distinct from laminin.

Brain Cells in Culture: Morphological Transformation by a Protein

One type of elmbryonic rat brain cell having an epithelioid morphology in the monolayer culture can be transformed by brain extract into cells having extensive processes resembling mature astrocytes. The transforming factor is a protein with a molecular weight of 350,000. A partially purified sample showed that it is active at a concentration as low as 1 x 10-8M. The transforming actvity is high in adult brains but low in embryonic brains and tumors of the nervous systems.

Early release of glia maturation factor and acidic fibroblast growth factor after rat brain injury

A major component of the healing response of the brain to injury is the induction of growth and trophic factors. In the rat brain, glia maturation factor (GMF) and acidic fibroblast growth factor (aFGF) are not extracellular. However, within the first hour following brain injury, the amount of GMF and aFGF in the wound cavity increased by 7- and 13-fold, respectively, compared to the tissue adjacent to the wound. A cascade of cellular and biochemical events, leading to glial proliferation, the arrest of secondary neuronal death and axonal sprouting, may be initiated by the sudden increase in the extracellular concentration of these factors.

Molecular Cloning and Expression of Biologically Active Human Glia Maturation Factor-β

Glia maturation factor‐β, a protein found in the brains of all vertebrates thus far examined, appears to play a role in the differentiation, maintenance, and regeneration of the nervous system. Using oligonucleotide probes based on the sequences of three tryptic peptides derived from bovine glia maturation factor‐β, we screened a human brainstem cDNA library in δll. A 0.7‐kb clone was isolated, sequenced in its entirety, and found to encode a polypeptide of 142 amino acids which contained regions identical to the three bovine peptides. This polypeptide, human recombinant glia maturation factor‐β, has been expressed in Escherichia coli and found to possess structural characteristics and biological activity indistinguishable from those of the native bovine protein.

Fine structure of cultured glioblasts before and after stimulation by a glia maturation factor.

Abstract The effect of Glia Maturation Factor on glioblasts in a monolayer culture is studied at the ultrastructural level. The most important finding consists of a change from cells with a predominance of sheath microfilaments (50 A) and desmosome junctions to cells with a predominance of 100 A-filaments (gliofilaments) and the puncta adhaerentia type of junctions. The restructuring of the cellular fine components, taken together with our earlier studies on chemistry and cytodynamics, is consistent with the maturation of glial cells in vitro. Although the microtubules do not alter in number, they change from a random orientiation to a parallel alignment with respect to the long axis of the cell processes. Glioblasts in aggregate cultures undergo morphological maturation without the help of the exogenous maturation factor. Glycogen granules characteristic of glia are also observed in these cells. The results suggest that the maturation factor is synthesized by the glial cells during the course of development and that cytodifferentiation is triggered by a local accumulation of the factor above a threshold level.

Multiple molecular forms of Glia maturation factor

Glia maturation factor from the pig brain can be detected in two molecular forms: the high molecular weight form which is 200 000 dalton in size and the low molecular weight form which is 40 000 dalton in size, as determined by Sephadex gel filtration. The former accounts for 85% of the total biological activity extracted at physiologic pH. The proportion of the low molecular weight form increases following freeze-thawing and ion-exchange chromatography. In addition to the morphological effects, both forms possess mitogenic activity but no esteropeptidase activity. Both forms show similar enzyme susceptibility, being inactivated by papain, ficin and pronase but resistant to subtilisin, thermolysin and trypsin. The high molecular weight form is more resistant to denaturation by low pH, heating and urea than the low molecular weight form. The high molecular weight factor has an isoelectric point of 4.27 whereas the low molecular weight factor has one of 5.04.

Glia maturation factor in bovine brain: Partial purification and physicochemical characterization

Glia maturation factor (GMF) is partially purified from bovine brains by the following procedure: extraction at physiologic pH, dialysis and freeze-drying of the extract, ethanol washing of the dried powder and re-extraction of the ethanol-washed residue with Tris-buffered saline, ion-exchange chromatography with DEAE Sephadex and molecular sieving with Bio-gel P-200. The partially purified protein has an apparent molecular weight of 23,000 and an isoelectric point of 4.75, and retains both morphological transforming and mitogenic activities when tested on glioblasts. Both activities are susceptible to protease digestion and heat inactivation. The procedure results in a 400-fold purification of the morphological activity and a 1400-fold purification of the mitogenic activity. Both activities are detectable when GMF is used in nanogram quantities. The possibility that both functions are expressions of the same factor and the possible role of GMF in the differential or sequential stimulation of cell growth and maturation are discussed.

Expression of Glia Maturation Factor β mRNA and Protein in Rat Organs and Cells

Abstract: Rat glia maturation factor β (GMF‐β) cDNA was obtained by reverse transcription of rat brain mRNA followed by polymerase chain reaction amplification, using primers from the human sequence. The deduced amino acid sequence of rat GMF‐β differed from the human counterpart in only three places: His27 in place of Asn, Val51 in place of Ile, and Leu93 in place of Val. The high degree of evolutionary conservation suggests that GMF‐β plays an essential role in animal cell physiology. The expression of GMF‐β mRNA in the rat was studied by the northern blot technique, using a rat cRNA probe corresponding to the entire coding region. GMF‐β mRNA was predominantly expressed in the brain and spinal cord, although trace levels were found in other organs, including testis and ovary. In the brain GMF‐β mRNA was detectable at as early as embryonic day 10, and persisted through as late as postnatal month 14, with minor variations in between. On the other hand, GMF‐β protein exhibited more obvious developmental changes, with its level increasing slowly prenatally and plateauing at 1 week after birth. GMF‐β mRNA and protein were also observed in several cultured cells. Some cells of neural origin contained higher levels of GMF‐β protein compared with cells derived from other sources. Through demonstration of mRNA and confirmation by immunoblotting, we conclude that GMF‐β is synthesized by rat organs and that GMF‐β is predominantly a brain protein.

Effects of glia maturation factor overexpression in primary astrocytes on MAP kinase activation, transcription factor activation, and neurotrophin secretion.

Using the replication-defective adenovirus vector, we overexpressed rat glia maturation factor (GMF) in primary astrocyte cultures derived from embryonic rat brains. Among the three isoforms of MAP kinase, there was a big increase in the phosphorylation of p38, as detected with Western blotting using the phosphospecific antibody. Likewise, there was a substantial increase in the phosphorylation of the transcription factor CREB. Using the electrophoretic mobility shift assay (EMSA), we found a stimulation in the transcription factor NF-κB. The activations of CREB and NF-κB were blocked by inhibitors of either p38 (SB-203580) or MEK (PD-098059), suggesting that they were events downstream of MAK kinase. There was an increased secretion of BDNF and NGF into the conditioned medium, along with an increase in their messenger RNA. The inductions of BDNF and NGF were also blocked by inhibitors of p38 and MEK, as well as by the inhibition of NF-κB with a decoy DNA sequence. Taken together, the results suggest that GMF functions intracellularly in astrocytes as a modulator of MAP kinase signal transduction, leading to a series of downstream events including CREB and NF-κB activation, resulting in the induction and secretion of the neurotrophins.