ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64030953.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Immunoglobulin heavy‐chain (IgH) gene expression is regulated largely by the IgH gene intronic enhancer (ENHiH), which is composed of multiple protein‐binding motifs. These motifs are DNA elements that are important for the regulation of IgH gene transcription. It has been reported that the HE2 (μB) and μA motifs within the ENHiHaffect B cell‐specific gene expression. To examine the function of the HE2 and μA elements in vivo, we established transgenic mouse lines. A deletion mutant of the human ENHiHthat contains the HE2 and μA motifs, but lacks the motifs corresponding to murine E5, E3, and octamer, functioned not only in B lymphocytes but also in choroid plexus cells, which secrete CSF. As a result, we obtained choroid plexus tumor‐bearing transgenic mice and could establish choroid plexus carcinoma cell lines. In addition, using the luciferase assay, we confirmed at the cellular level that the HE2 motif shows a fair degree of enhancer activity in cultured choroid plexus carcinoma cells. These results suggest that existence of atrans‐acting factor for the HE2 motif in choroid plexus cells. Actually, in this cultured cell line, the existence of a protein binding to the HE2 motif was demonstrated by a gel retardation assay. Due to the sequence homology between the HE2 motif and the Ets‐binding sites, an Ets‐related protein is a highly probable candidate for being t

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64030961.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We have created transgenic mice bearing varying copy numbers of a transgene coding for normal DM‐20, the alternatively spliced quantitatively minor isoform of myelin proteolipid protein. Demyelination of the CNS occurs as a consequence of 70 copies of this transgene. Overt symptoms begin at ∼3 months with a wobbling gait. Occasional seizures lasting a few seconds begin at 3–4 months. These symptoms progress in severity with age. Death occurs by 8–10 months. Myelination in 2‐month‐old animals, before the onset of any overt symptoms, appears morphologically normal at the electron microscopic level. However, the myelin in these 2‐month‐old animals has a reduced amount of the major myelin proteolipid protein and about three times as much DM‐20 as normal animals. In 7‐month‐old animals that appear to be undergoing demyelination in the CNS, both the major myelin proteolipid protein and DM‐20 are greatly reduced relative to the 2‐month‐old animal. Mice with 17 copies of the transgene also have a reduced amount of the major myelin proteolipid protein but appear to be otherwise normal and have normal life spans (>2 yr). Mice with low copy numbers of the transgene (2–4 copies) appear to be unaffect

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64030967.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:A cDNA clone encoding a human γ‐aminobutyric acid (GABA) transporter has been isolated from a brain cDNA library, and its functional properties have been examined in mammalian cells. The nucleotide sequence predicts a transporter with 614 amino acids and 12 putative transmembrane domains. The highest degree of amino acid identity is with a betaine/GABA transporter originally cloned from the dog termed BGT‐1 (91%) and a related transporter from mouse brain (87%). These identities are similar to those for species homologues of other neurotransmitter transporters and suggest that the new clone represents the human homologue of BGT‐1. The transporter displays high affinity for GABA (IC50of 30 µM) and is also sensitive to phloretin,l‐2,4‐diaminobutyric acid, and hypotaurine (IC50values of ∼150–400 µM). The osmolyte betaine is ∼25‐fold weaker than GABA, displaying an IC50of ∼1 mM. The relative potencies of these inhibitors at human BGT‐1 differ from those of mouse and dog BGT‐1. Northern blot analysis reveals that BGT‐1 mRNA is widely distributed throughout the human brain. The cloning of the human homologue of BGT‐1 will further our understanding of the roles of GABA

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64030977.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Choline acetyltransferase activity is barely detectable in a mutant pheochromocytoma PC12 cell line, A123.7, which is deficient in protein kinase A activity. Northern blot and polymerase chain reaction analyses showed that this mutant cell line has dramatically reduced levels of choline acetyltransferase mRNA, which correlates with the low level of enzyme activity. Transient transfection analysis was used to assess the functionality, in these cells, of an enhancer element and a cholinergic‐specific repressor element derived from the human choline acetyltransferase gene. The results show that the enhancer element is inactive in the protein kinase A‐deficient cell line. Cotransfection experiments with plasmids expressing the catalytic subunit of protein kinase A support this conclusion. These data indicate that protein kinase A regulates expression of the choline acetyltransferase gene at the transcriptional level by controlling the activity of an enhancer elem

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64030985.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:In brain, astrocytes and endothelial cells are a major site of adenosine degradation. These two cell types, found in close apposition, constitute the wall of the brain's capillaries and serve as a site of hypoxanthine production and degradation. Both cell types possess the hypoxanthine salvage pathway and can incorporate hypoxanthine into nucleotides. This suggests that the endothelial‐astrocyte anatomical complex might play an important role in the brain's purine homeostasis. To test this hypothesis, cocultures of monolayers of vascular endothelial cells and astrocytes were grown over a porous membrane, in close apposition to one another, and studies on hypoxanthine transport and metabolism to uric acid were performed. The flux of hypoxanthine across the cell layers was simultaneously determined and compared with the flux of sucrose, as a probe of passive diffusion. Our results show that in endothelial, glial, and endothelial‐glial cell layers the hypoxanthine flux was greater than that of sucrose, and that the flux of hypoxanthine, but not of sucrose, was inhibited by adenine or by lowering the temperature. These results suggest that hypoxanthine moves across endothelial, glial, and endothelial‐glial cell layers by a transport process. Furthermore, we found that hypoxanthine transport is enhanced when glial and endothelial cells are cocultured compared with that in glial or endothelial monolayers. In addition the coculture also resulted in a depression of xanthine oxidase act

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64030991.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Okadaic acid (OA) is a frequently used phosphatase inhibitor that by inhibiting dephosphorylation increases the net phosphorylation level in various systems. In the present study OA was used to assess the role of balanced phosphorylation‐dephosphorylation reactions for successful regeneration of peripheral nerves. To achieve this, the effects of OA on phosphorylation levels, neurite outgrowth, injury‐induced support cell proliferation, and neurofilament stability, respectively, were investigated in the in vitro regenerating, adult frog sciatic sensory nerve. OA at a moderate concentration (20 nM) increased phosphorylation levels and almost completely inhibited the in vitro regeneration in a reversible way. The effect on regeneration was not due to induced neurofilament instability and was only seen when the drug was applied in the outgrowth region. The latter and the absence of effects on support cell proliferation indicate that OA acts locally at the level of newly formed axons. However, the inhibition of regeneration was not a consequence of reduced delivery of proteins by axonal transport, because this process in fact was increased by OA. Altogether, the study suggests that properly balanced phosphorylating‐dephosphorylating reactions are critical for regeneration of peripheral n

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64031000.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:Endothelin‐1 (ET‐1), originally characterized as a potent vasoconstrictor peptide secreted by vascular endothelial cells, has now been described to possess a wide range of biological activities within the cardiovascular system and in other organs. Brain microvessel endothelial cells, which, together with perivascular astrocytes, constitute the blood‐brain barrier, have been shown to secrete ET‐1, whereas specific ET‐1 receptors are expressed on astrocytes. It is reported here that conditioned medium from primary cultures of mouse embryo astrocytes could significantly, and reversibly, attenuate the accumulation of both ET‐1 and its precursor big ET‐1 in the supernatant of rat brain microvessel endothelial cells by up to 59 and 76%, respectively, as assessed by immunometric assay. This inhibitor of ET‐1 production was purified by gel‐exclusion and ion‐exchange chromatography as a 280‐Da iron‐containing molecule, able to release nitrites upon degradation. These results suggest that astrocytes, via release of an iron‐nitrogen oxide complex, may be involved in a regulatory loop of ET‐1 production at the level

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64031008.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:PEA‐15 has recently been identified as a major phosphoprotein in astrocytes and an endogenous substrate for protein kinase C. This 15‐kDa protein exists under three molecular forms, an unphosphorylated form, N, and two phosphorylated forms, Pa and Pb. Ȧntisera were raised against synthetic peptides corresponding to the internal sequences of the mouse protein containing the two specific phosphorylation sites and affinity‐purified antibodies were used for immunoblotting. PEA‐15 was found mainly in the cytosol, but its protein kinase C‐phosphorylated form, Pb, was also detectable in association with the membrane and remained with the fraction that contains stabilized microtubules. Abundant in astrocytes, particularly in the hippocampus, PEA‐15 was also detected in all cultured brain cell types examined, indicating a more ubiquitous distribution of the protein, further demonstrated by its detection in the eye and in the lung. Parallel to the increase in expression levels, phosphorylation of PEA‐15 also increased during development. This paralleled results obtained in primary cultures, where PEA‐15 levels increase with cell maturation. Finally, physiological importance of PEA‐15 phosphorylation was illustrated by immunoreactivity observed in brain homogenates of different mammals, birds, am

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64031016.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY

摘要:

Abstract:We studied by immunocytochemical localization, the glycine neurotransmitter transporter (GLYT2) in mouse brain, using polyclonal antibodies raised against recombinant N‐terminus and loop fusion proteins. Western analysis and immunocytochemistry of mouse brain frozen sections revealed caudal‐rostral gradient of GLYT2 distribution with massive accumulation in the spinal cord, brainstem, and less in the cerebellum. Immunoreactivity was detected in processes with varicosities but not cell bodies. A correlation was observed between the pattern we obtained and previously reported strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission accompanying the glycine receptor at the classic inhibitory system in the hindbr

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1995.64031026.x

出版商:Blackwell Science Ltd    

年代:1995

数据来源: WILEY