Expression Of MAG Research Articles

Efficacy of genetically engineered oncolytic virus on glioma stem cells in vitro

Objective Exploring the effect of endostatin and angiostatin(Endo-Angio) fusion genemodified herpes simplex virus ( VAE ) on glioma stem cells ( GSCs ) in vitro. Method Surgical specimens of human high grade glioma ( WHO Ⅲ, Ⅵ) were collected, and GSCs were isolated under the culture conditionsoriginally designed for selective expansion of neural stem cells. In order to identify GSCs,the number of CD133 positive cells in GSCs were detected by flow cytometry; self - renewal capacity and the expression of Nestin, GFAP, NF, and MAG of GSCs were detected by monoclonal forming and immunofluorescence assay respectively. After that, GSCs viability was detected by MTS assay. Expression of Endo - Angio fusion gene at mRNA and protein level was detected by RT - PCR and Western blot. At last,the efficacy of fusion protein to human brain microvascular endothelial cells (HBMEC) and the biological properties of GSCs were detect after infected by VAE. Results ( 1 )4 GSCs isolated from 20 surgical specimens could suspend growth and had the ability of self-renewal and multipotential differentiation,and could express neural stem cells marker,CD133 and Nestin. (2)VAE could infect GSCs and significantly inhibit the viability of GSCs( P ＜0.05). (3) Significant expression of Endo-Angio fusion gene was observed and it could inhibit HBMEC proliferation( P ＜0.05). (4) Residual viable cells lost the ability of self - renewal and adherent differentiation. Conclusions In vitro, VAE can inhibit the activity of GSCs and the expression of exogenous Endo - Angio fusion gene can inhibit HBMEC proliferation. VAE can be used as a novd virus -gene therapy strategy for glioma. Key words: Glioma; Neoplastic stem cells; Endostatins; Angiogenesis inhibitors; Virus - gene therapy

Studies on functional role of DNA methylation within the FXYD5-COX7A1 region of human chromosome 19

We used the Rapid Identification of Genomic Splits technique to get a detailed methylation landscape of a 1-megabase-long human genome region (FXYD5-COX7A1, chromosome 19) in normal and tumor lung tissues and in the A549 lung cancer cell line. All three samples were characterized by an essentially uneven density of unmethylated sites along the fragment. Strikingly enough, the distribution of hypomethylated regions did not correlate with gene locations within the fragment. We also demonstrated that the methylation pattern of this long genomic DNA fragment was rather stable and practically unchanged in human lung cancer tissue as compared with its normal counterpart. On the other hand, the methylation landscape obtained for the A549 cell line (human lung carcinoma) in the USF2-MAG locus showed clear differences from that of the tissues mentioned above. A comparative analysis of transcriptional activity of the genes in this region demonstrated the general absence of direct correlation between methylation and expression, although some data suggest a possible role of methylation in the regulation of MAG expression through cis-regulatory elements. In total, our data provide new evidence for the necessity of revising currently prevailing views on the functional significance of methyl groups in genomic DNA.

중추신경계 손상 회복에 미치는 대한 조구등의 영향

Objective : Following central nervous system(CNS) injury, inhibitory influences at the site of axonal damage occur. Glial cells become reactive and form a glial scar, gliosis. Also myelin debris such as MAG inhibits axonal regeneration. Astrocyte-rich gliosis relates with up-regulation of GFAP and CD81, and eventually becomes physical and mechanical barrier to axonal regeneration. MAG is one of several endogenous axon regeneration inhibitors that limit recovery from CNS injury and disease. It was reported that molecules that block such inhibitors enhanced axon regeneration and functional recovery. Recently it was reported that treatment with anti-CD81 antibodies enhanced functional recovery in the rat with spinal cord injury. So in this current study, the author investigated the effect of the water extract of Uncariae Ramulus et Uncus on the regulation of CD81, GFAP and MAG that increase when gliosis occurs. Methods : MTT assay was performed to examine cell viability, and cell-based ELISA, western blot and PCR were used to detect the expression of CD81, GFAP and MAG. Then also immunohisto- chemistry was performed to confirm in vivo. Results : Water extract of Uncariae Ramulus et Uncus showed relatively high cell viability at the con- centration of 0.05%, 0.1% and 0.5%. The expression of CD81, GFAP and MAG in astrocytes was decreased after the administration of Uncariae Ramulus et Uncus water extract. These results was confirmed in the brain sections following cortical stab injury by immunohistochemistry. Conclusion : The authors observed that Uncariae Ramulus et Uncus significantly down-regulates the expression of CD81, GFAP and MAG. These results suggest that Uncariae Ramulus et Uncus can be a candidate to regenerate CNS injury.

Oligodendroglial abnormalities in schizophrenia, mood disorders and substance abuse. Comorbidity, shared traits, or molecular phenocopies?

The evidence implicating oligodendroglia in major mental disorders has grown significantly in the past few years. Microarray analysis revealed altered expression of oligodendroglia-related genes in multiple brain regions from several, clinically diverse groups of subjects with schizophrenia (SZ) as well as subjects with bipolar disorder (BD) and major depressive disorders (MDD), alcoholics and cocaine users. In line with gene expression findings, evidence for ultrastructural changes in white matter and altered oligodendroglia in these disorders were reported in neuroimaging and neuropathological studies. Changes in oligodendroglia-related genes reported in SZ, BD and MDD appear to display considerable similarities (particularly decreased expression of MAG, ERBB, TF, PLP1, MOG, MOBP, MOG), while changes in cocaine abuse and alcoholism are more diverse. Common oligodendroglial abnormalities might indicate aetiological or pathophysiological overlaps between different disorders. The possible mechanisms of oligodendroglial abnormalities may involve functional variations in oligodendroglia-related genes, epigenetic regulation of chromatin, DA system hyperactivity and other mechanisms.

Expression of MBP, PLP, MAG, CNP, and GFAP in the Human Alcoholic Brain

Chronic and excessive alcohol misuse results in neuropathological damage in the cerebral cortex. The damage includes white matter loss, brain atrophy, and selective loss of neurons in the superior frontal gyrus. Chronic alcohol misuse also results in alterations in the expression of a number of genes, including a selective reprogramming of myelin gene expression in the frontal cortex. The expression of cyclic nucleotide phosphodiesterase, glial fibrillary acidic protein, myelin-associated glycoprotein, myelin basic protein, and myelin proteolipid protein were assessed in the superior frontal gyrus and the primary motor cortex of control, uncomplicated alcoholic, and cirrhotic alcoholic cases. Overall, the expression of cyclic nucleotide phosphodiesterase, glial fibrillary acidic protein, myelin-associated glycoprotein, and myelin basic protein were significantly lower in the cirrhotic alcoholic cases compared with controls, with a similar tendency for myelin proteolipid protein. There was a strong correlation between the expression of the proteins studied and the brain weight of the individual case, but this interaction did not confound the overall analysis. There was no significant difference between controls and uncomplicated alcoholics. The loss of myelin proteins occurred without gross changes in brain pathology or brain weight and was not restricted to pathologically susceptible brain regions. It is not possible to determine whether the loss of myelin proteins in cirrhotic alcoholics is the result of cirrhosis per se or the combination of alcohol misuse and liver cirrhosis. Future studies comparing cases with alcoholic and nonalcoholic cirrhosis of the liver disease are required to elucidate this further.

Myelin-associated glycoprotein (Siglec-4) expression is progressively and selectively decreased in the brains of mice lacking complex gangliosides.

Myelin-associated glycoprotein (MAG, Siglec-4) is a quantitatively minor membrane component expressed preferentially on the innermost myelin wrap, adjacent to the axon. It stabilizes myelin-axon interactions by binding to complementary ligands on the axolemma. MAG, a member of the Siglec family of sialic acid-binding lectins, binds specifically to gangliosides GD1a and GT1b, which are the major sialoglycoconjugates on mammalian axons. Mice with a disrupted Galgt1 gene lack UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and fail to express complex brain gangliosides, including GD1a and GT1b, instead expressing a comparable amount of the simpler gangliosides GM3, GD3, and O-acetyl-GD3. Galgt1-null mice produce similar amounts of total myelin compared to wild-type mice, but as the mice age, they exhibit axon degeneration and dysmyelination with accompanying motor behavioral deficits. Here we report that Galgt1-null mice display progressive and selective loss of MAG from the brain. At 1.5 months of age, MAG expression was similar in Galgt1-null and wild-type mice. However, by 6 months of age MAG was decreased approximately 60% and at 12 months of age approximately 70% in Galgt1-null mice compared to wild-type littermates. Expression of the major myelin proteins (myelin basic protein and proteolipid protein) was not reduced in Galgt1-null mice compared to wild type. MAG mRNA expression was the same in 12-month-old Galgt1-null compared to wild-type mice, an age at which MAG protein expression was markedly reduced. We conclude that the maintenance of MAG protein levels depends on the presence of complex gangliosides, perhaps due to enhanced stability when MAG on myelin binds to its complementary ligands, GD1a and GT1b, on the apposing axon surface.

Corticoids protect oligodentrocyte precursor cells against cytokine-induced damage

In preterm fetuses an ascending intrauterine inflammation before or during birth can be associated with periventricular leukomalacia (PVL). PVL is characterised by a disrupted. myelination [corrected] due to a loss of oligodendrocyte progenitors. Inflammatory cytokines not only induce apoptotic cell death but in addition prevent the differentiation of the immature A2B5 oligodendrocytc progenitors into the myelinating phenotype. The aim of the present study was to clarify whether corticosteroids can protect oligodendrocyte progenitors from such injury. Cultures [corrected] of more than 90 % A2B5 positive progenitors were prepared from neonatal sprague dawley rats. After 1 day in proliferation medium, cells were treated with interferon-gamma [corrected] (IFN-gamma; 10 U/ml) and tumour necrosis factor-alpha [corrected] (TNF-alpha; 10 ng/ml) for 48 h. In addition, corticosterone (C), deoxycorticosterone (DC), or dexamethasone (DEX) (1 micro M) were applied to the incubation medium of the study groups. After treatment cultures were transferred to media containing factors to promote differentiation of progenitors into thc myelinating phenotype. At culture day 9 total cell [corrected] number was determined. In addition, cells were analysed by immunocytochemistry for A2B5 as well. as by RT-PCR [corrected] and western-blot-analysis of the [corrected] myelin-specific proteins [corrected] MBP, MAG and CNP. Application of INF-gamma and TNF-alpha caused a dramatic decrease in cell number culture day 9 [corrected] More than 30 % of the surviving cells were A2B5-positive compared to 1 % in control dishes. Protein expression of MBP, MAG and CNP as well as mRNA expression, of MAG and MBP were considerably reduced after administration of INF- and TNF-. Co-treatment with, corticosteroids significantly increased total cell count, dexamethasone showing the strongest protective effect.Moreover, corticosteroids alleviated the cytokine induced inhibition in protein- and mRNA-expression [corrected] of MBP, MAG and CNP. Corticosteroids therefore protect oligodendrocyte progenitors from cytokine-induced cell damage.

Characterization of three human oligodendroglial cell lines as a model to study oligodendrocyte injury: morphology and oligodendrocyte-specific gene expression.

Oligodendrocytes, the myelin-forming cells of the central nervous system, are the target of pathogenic immune responses in multiple sclerosis. Primary cultures of human oligodendrocytes have been used to unravel the cellular and molecular mechanisms of immune-mediated injury of oligodendrocytes. However, these studies are hampered by the limited availability of viable human brain tissue. The present study was aimed at comparing the morphological and biochemical characteristics of the human oligodendroglial cell lines HOG, MO3.13 and KG-1C. We have determined oligodendrocyte-associated features of these lines and analyzed the degree to which they can be used as a model of human oligodendrocytes arrested at specific developmental stages. The oligodendroglial cell lines all exhibited markers of immature oligodendrocytes, such as CNPase and GalC, but not the astrocytic marker GFAP. Differentiation could be induced in HOG and MO3.13 cells, as was seen through a decrease in proliferation, an increase in process extension without formation of myelin sheets and up-regulation of genes associated with mature oligodendrocytes such as MBP and MOG. Microarray analysis revealed the expression of MAG, MOBP and OMG genes in HOG cells. The KG-1C cells displayed poor growth characteristics in the recommended conditions. In conclusion, our data show that the oligodendroglial cell lines HOG and MO3.13 can be used as a model of human oligodendrocytes "arrested" in an immature developmental stage. Culturing in appropriate medium can induce further differentiation of these cells. These cell lines can therefore be applied as a model to study immune-mediated injury of oligodendrocytes in relation to disease.

CNS myelinogenesis in vitro: myelin basic protein deficient shiverer oligodendrocytes.

The shiverer mutant mouse is an autosomal recessive mutant characterized by incomplete myelin sheath formation in the central nervous system (CNS). Such mice contain a deletion in the MBP gene, do not produce MBP proteins, and have little or no compact myelin in the CNS. To investigate the myelin sheath formation in shiverer mutant mice resulting from the absence of compact myelin, firstly we developed new methods for generating oligodendrocyte precursor cells (OPCs) from an E17 mouse brain, and examined homozygous shiverer (shi/shi) OPCs with respect to myelinogenesis in vitro. After treatment of shi/shi OPCs in vitro with PDGF or bFGF, proliferation of shi/shi OPCs was enhanced similar to that observed in wild-type OPCs. The majority of cells from the shiverer mutant mouse, however, remained as A2B5-immunoreactive early OPCs. To determine which molecular events affect the differentiation of shi/shi OPCs, we determined the signaling pathway that could be responsible for activating myelin sheath-specific proteins. We found that the developmental schedule of shi/shi OPCs in vitro was accelerated by the addition of cyclic AMP analogs, dibutyryl cAMP (dbcAMP). Treatment of shi/shi OPCs with dbcAMP had significant effect on the differentiation of OPCs that became MAG-expressing oligodendrocytes. To further determine the possible mechanism involved in the activation of MAG by dbcAMP, we examined the cAMP-dependent signaling cascades. The activation of JNK was markedly stimulated by treatment with dbcAMP, and the phosphorylation of transcription factor ATF-2 was also stimulated by dbcAMP. We demonstrated that the MAG-positive shi/shi oligodendrocytes extend processes around axons and finally covered the axon, this was clearly observed by immunocytochemistry of shi/shi oligodendrocyte-DRG cocultures. These results suggest that ATF-2 coupled to specific signal transduction cascades plays an important regulatory role in MAG expression at a specific stage of shi/shi oligodendrocyte differentiation, and OPCs grow to become myelin-forming cells with numerous cell processes that wraps around an axon to form a thin myelin sheath.

Effects of macrophage transplantation in the injured adult rat spinal cord: a combined immunocytochemical and biochemical study.

Early and robust invasion by macrophages may be one of the reasons why axonal regeneration is more effective in the PNS than in the CNS. Therefore, we have grafted autologous peritoneal macrophages labeled with fluorescent latex microspheres into spinal cord compression lesions. At various survival times, we have studied their effect on the expression of neuronal (neurofilaments [NF], calcitonin gene-related peptide [CGRP], 5-hydroxytryptamine [5-HT]) and nonneuronal markers (myelin-associated glycoprotein [MAG], glial fibrillary acidic protein [GFAP], laminin) by using semiquantitative Western blot and immunohistochemical techniques. After 1 month, we observed a significant decrease of the expression of MAG as well as an important invasion of the lesion site by neurites, chiefly peptidergic axons of presumed dorsal root origin, in macrophage-grafted animals compared with controls. In addition, angiogenesis and Schwann cell infiltration were more pronounced after macrophage grafts, providing an increase in laminin, a favorable substrate for axonal regrowth. By using reverse transcription-polymerase chain reaction (RT-PCR), mRNAs for tumor necrosis factor-alpha (TNF-alpha) were detected in the transplanted cells, whereas results were negative for nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), or acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Thus, macrophage grafts may represent an interesting strategy to promote axonal regeneration in the CNS. Our study suggests that they may exert their beneficial effects by degrading myelin products, which inhibit axonal regrowth, and by promoting a permissive extracellular matrix containing notably laminin. No evidence for a direct synthesis of neurotrophic factors by the transplanted macrophages was found in this study, but resident glial cells could secrete such factors as a result of stimulation by macrophage-released cytokines.

Differential expression of MAG, MBP and L1 in the developing lateral superior olive

Differential expression of MAG, MBP and L1 in the developing lateral superior olive

Purification and properties of the alkylation repair DNA glycosylase encoded the MAG gene from Saccharomyces cerevisiae.

The MAG gene of Saccharomyces cerevisiae encodes an alkylation repair DNA glycosylase whose sequence is homologous to the AlkA DNA glycosylase from Escherichia coli. To investigate the biochemical properties of MAG in comparison to AlkA, MAG was expressed in E. coli and purified to electrophoretic homogeneity. N-Terminal sequencing of the purified protein identified the translational start site which corresponded to that predicted previously from the nucleotide sequence. Polyclonal antibodies raised against MAG inhibited the enzymatic activity of MAG, but not that of AlkA, and vice versa, implying that the structures of the active site regions of these enzymes are antigenic, but sufficiently different to have different epitopes. Kinetic analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea and [3H]dimethyl sulfate showed that MAG was as effective as AlkA in removing 3-methyladenine, 7-methylguanine, and 7-methyladenine. However, the purified MAG enzyme did not catalyze the excision of O2-methylthymine, which is a major substrate for AlkA. Furthermore, 3-methylguanine was excised 20-40 times more slowly by MAG than by AlkA. The kinetics of 3-methylguanine excision by MAG were found to be similar to the low rate of 3-methylguanine excision catalyzed by 3-methyladenine DNA glycosylase I (Tag) of E. coli. Expression of MAG in alkA mutant cells did not effectively restore alkylation resistance of the mutant as did AlkA itself. It thus appears that MAG is a less versatile enzyme than AlkA in spite of the sequence relationship and may have a similar function in yeast as the nonhomologous Tag enzyme in E. coli.

Protection against chloroethylnitrosourea cytotoxicity by eukaryotic 3-methyladenine DNA glycosylase.

A eukaryotic 3-methyladenine DNA glycosylase gene, the Saccharomyces cerevisiae MAG gene, was shown to prevent N-(2-chloroethyl)-N-nitrosourea toxicity. Disruption of the MAG gene by insertion of the URA3 gene increased the sensitivity of S. cerevisiae cells to N-(2-chloroethyl)-N-nitrosourea, and the expression of MAG in glycosylase-deficient Escherichia coli cells protected against the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea. Extracts of E. coli cells that contain and express the MAG gene released 7-hydroxyethylguanine and 7-chloroethylguanine from N-(2-chloroethyl)-N-nitrosourea-modified DNA in a protein- and time-dependent manner. The ability of a eukaryotic glycosylase to protect cells from the cytotoxic effects of a haloethylnitrosourea and to release N-(2-chloroethyl)-N-nitrosourea-induced DNA modifications suggests that mammalian glycosylases may play a role in the resistance of tumor cells to the antitumor effects of the haloethylnitrosoureas.

A common element involved in transcriptional regulation of two DNA alkylation repair genes (MAG and MGT1) of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents. MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA. We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression. Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS. One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S. cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase. Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements. We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S. cerevisiae DNA repair genes.

A common element involved in transcriptional regulation of two DNA alkylation repair genes (MAG and MGT1) of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents. MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA. We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression. Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS. One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S. cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase. Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements. We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S. cerevisiae DNA repair genes.

Induction ofS.cerevisiae MAG3-methyladenine DNA glycosylase transcript levels in response to DNA damage

We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E. coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents. Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide. Unlike some other yeast DNA-damage-inducible genes, MAG expression is not induced by heat shock. The S. cerevisiae MGT1 O6-methylguanine DNA methyltransferase is not involved in regulating MAG gene expression since MAG is efficiently induced in a methyltransferase deficient strain; similarly, MAG glycosylase deficient strains and four other methylmethane sulfonate sensitive strains were normal for alkylation-induced MAG gene expression. However, de novo protein synthesis is required to elevate MAG mRNA levels because MAG induction was abolished in the presence of cycloheximide. MAG mRNA levels were equally well induced in cycling and G1-arrested cells, suggesting that MAG induction is not simply due to a redistribution of cells into a part of the cell cycle which happens to express MAG at high levels, and that the inhibition of DNA synthesis does not act as the inducing signal.