ABSTRACT
Human glia maturation factor-gamma (hGMFG) was recently identified as a gene that is homologous to glia maturation factor-beta (GMFB). In this study, we determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has putative elements for binding transcription factors Sp-1, GATA-1, AML-1a, Lyf-1 and Ets-1, but there were no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region -84 to -70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We found that 226 bp of the core promoter region exhibited bidirectional promoter activity.
Subject(s)
Glia Maturation Factor/genetics , Promoter Regions, Genetic/physiology , Base Sequence , Binding Sites , Cell Line , Cloning, Molecular , Genes, Regulator , Glia Maturation Factor/biosynthesis , Humans , Molecular Sequence Data , Transcription Initiation SiteABSTRACT
We examined the effects on astrocytes of ACTH, which is used to treat West syndrome. We stimulated cultured rat astrocytes with ACTH(1-24), corticotropin-releasing factor, and dexamethasone, and examined changes in neurotrophic factor mRNAs by reverse transcription-PCR. Down-regulation of ciliary neurotrophic factor mRNA expression was observed by stimulation with ACTH(1-24), but the expression of nerve growth factor, brain-derived neurotrophic factor, and nerotrophin-3 mRNAs was unaffected. Northern blot analysis revealed that the decrease in ciliary neurotrophic factor mRNA occurred 4 h after stimulation with more than 10 nM of ACTH(1-24). Up-regulation of nerotrophin-3 mRNA expression was found after stimulation with 1 mM dexamethasone. These results suggest that ACTH(1-24) administrated in West syndrome may influence the expression of neurotrophic factors in astrocytes in vivo.
Subject(s)
Astrocytes/drug effects , Astrocytes/metabolism , Ciliary Neurotrophic Factor/genetics , Cosyntropin/pharmacology , Animals , Base Sequence , Brain-Derived Neurotrophic Factor/genetics , Cells, Cultured , Corticotropin-Releasing Hormone/pharmacology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , DNA/genetics , Dexamethasone/pharmacology , Down-Regulation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Infant , Nerve Growth Factor/genetics , Neurotrophin 3/genetics , Protein Kinase C/antagonists & inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Spasms, Infantile/drug therapy , Spasms, Infantile/metabolismABSTRACT
Serine proteases are known to be involved in neural development and various functions in the central nervous system. Mouse brain serine proteinase (mBSP) is expressed almost exclusively in the mouse brain and it has been characterized at the molecular and biochemical levels. In this study, we analyzed the developmental changes and localization of mBSP mRNA and protein in the mouse brain, using reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemistry. Expression of mBSP was strong in the white matter and the nerve tracts after postnatal day 30, especially in the cerebellum and the medulla oblongata. These results suggest that mBSP contributes to development and sustaining the functions in the mouse brain.
Subject(s)
Brain Chemistry/genetics , Brain/enzymology , Nerve Tissue Proteins/metabolism , RNA, Messenger/metabolism , Serine Endopeptidases/metabolism , Animals , Brain/embryology , Brain/growth & development , Embryo, Mammalian , Mice , Mice, Inbred ICR , Nerve Tissue Proteins/genetics , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Serine Endopeptidases/analysis , Serine Endopeptidases/geneticsABSTRACT
We have previously reported the unique heat sensitivity of a cell line of malignant fibrous histiocytoma cells, the MFH-2NR cell line. In the present study, treatment of MFH-2NR cells, at 43 degrees C for 1 h evoked typical apoptosis in these cells, which showed characteristic morphological changes, such as internucleosomal DNA fragmentation (DNA ladders), cell shrinkage, and chromatin condensation. Under these conditions, we examined p53 and bax protein levels, and p53 and bax mRNA expression to assess the potential relationship between these two proteins for the induction of apoptosis. The p53 protein, which is usually detected in trace amounts in normal cells, was highly expressed in untreated MFH-2NR cells, and the level did not increase after heat treatment, whereas the bax protein level increased from 30 min after the treatment. No change in p53 mRNA was found, but a transient increase in bax mRNA, peaking at 30 min, was detected by Northern blotting. DNA sequence analysis of the p53 gene from MFH-2NR cells demonstrated a GGG right arrow GAG homozygous point mutation in codon 242 of exon 6. These results suggest that the expression of bax protein and mRNA was augmented by a p53-independent pathway in the hyperthermia-induced apoptosis of MFH-2NR cells.
