摘要:

Abstract:Acetylcholinesterase (AChE), a highly conserved enzyme in the animal kingdom, is distributed throughout a wide range of vertebrate tissues where it is expressed as multiple molecular forms comprising different arrangements of catalytic and structural subunits. The major AChE form in the CNS is an amphiphilic globular tetramer (G4AChE) consisting of four identical catalytic subunits attached to cellular membranes by a hydrophobic noncatalytic subunit (P‐subunit). This study focuses primarily on current data involving the structure of the G4AChE P‐subunit, the expression and regulation of G4AChE during development and adulthood, and its role(s) in certain neurological disorders including Alzheimer's dise

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041335.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Levels of the GABAAreceptor α1‐, α6‐, β2‐, β3‐, γ2‐, and δ‐subunit mRNAs in cerebellar granule neurons rise concurrently during the second week of postnatal ontogeny. Previous studies in culture have suggested that extrinsic signals control these increases, but little is known about the nature of the regulatory cues. To determine when granule neurons become competent to express these six subunit mRNAs in mature patterns and to gain insight into their regulation, reverse transcriptase‐PCR was used to examine transcript expression in cultured granule neurons prepared at 2‐day intervals from postnatal days 2 through 10. Although only one pattern of expression was observed in vivo, three patterns were detected in culture. First, the levels of the α1‐ and α6‐subunit mRNAs were constant in cultures prepared at all ages. Second, the levels of the β2‐, β3‐, and γ2‐subunit mRNAs were constant in cultures prepared at postnatal days 2–6 but increased in those prepared at days 8–10. Third, the δ‐subunit mRNA level increased over time in culture regardless of cerebellar age at plating. Moreover, only δ‐subunit transcript expression was modulated by cell density. These findings indicate that the subunit transcripts are different

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041347.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Nuclear factor I (NFI) binding sites are present in a wide range of brain‐specific gene enhancer and promoter sequences and appear to play a role in establishing cell type‐specific expression within the CNS. The precise mechanisms used by various members of the NFI family of proteins to confer brain‐specific expression are unclear. We have addressed this issue by comparing the transactivating capabilities of two forms of NFI in directing gliotropic expression from two different JC virus (JCV) promoter configurations. The JCV is an opportunistic pathogen of humans that causes lytic destruction of the oligodendrocytes and thus demyelination in immunocompromised patients. Our results show that the cerebellum‐enriched form of NFI (NFI‐A1) transactivates two gliotropic JCV early promoters to a greater extent than the ubiquitous form of NFI (NFI‐C1). Activation by NFI‐A1 was dramatically greater in glial than in nonglial cells. These results suggest that NFI proteins direct brain‐specific expression through combinatorial interactions with cell specific coactivators and/or transcription factors that recognize adjacent sites within brain spe

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041354.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The ability of the neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), and neurotrophin‐4/5 (NT‐4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron‐specific enolase‐immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT‐4/5, or NT‐3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT‐4/5 (46%), whereas NT‐3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT‐3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT‐4/5, and NT‐3, but not NGF, induced the rapid expression of the immediate early gene c‐fos. Immunocytochemical double‐labeling with antibodies to c‐fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c‐fos. After 4 days in vitro, both BDNF and NT‐3 induced the formation of c‐fos protein in calbindin‐immunopositive neurons, whereas NT‐4/5 did not. The latter results suggest that although BDNF and NT‐4/5 have been shown to act through a common receptor, TrkB, it appears that t

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041362.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:β‐Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that β‐amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of β‐amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 nM, thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of β1–42 and β25–35 peptides. In cultured astrocytes, the stellate morphology induced by β1–42 and β25–35 aggregates can be prevented and reversed by thrombin exposures between 10 pMand 1 µM. In contrast, thrombin potentiates rather than attenuates the β‐amyloid‐induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of β‐amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor‐activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin‐1. These data provide both new insight into the signaling pathways underlying the cellular effects of β‐amyloid and additional support for the role of thrombin as an important

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041374.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We previously demonstrated that the inhibitory neurotransmitter glycine induced membrane currents in glial cells from rat spinal cord. In the present study, the patch‐clamp technique was combined with the reverse transcription‐mediated PCR to analyze the glycine receptor‐subunit expression in individual glial cells of rats age 3–18 days. Using the patch‐clamp technique in the whole‐cell configuration, glial cells were identified by their membrane current pattern and tested for responsiveness to glycine. Subsequently, the cytoplasm was harvested followed by reverse transcription of total cytoplasmic RNA. Subunit‐specific cDNA fragments were amplified and analyzed by agarose gel electrophoresis, Southern blotting, and sequencing. In all glial cell types investigated, transcripts of the α1 subunit, but not of α2 or α3 subunits, were detected. In addition, about one‐half the glial cells analyzed contained β‐subunit mRNA. These results illustrate that glial cells of rat spinal cord express functional glycine receptors in contrast to cultured glial cells. Glial cells are in intimate contact with synaptic regions making it likely that these nonneuronal receptors may be activated during glycinergic transmission and may trigger yet unknown respons

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041383.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin‐6‐receptor transducer as well as gp190/low‐affinity LIF receptor. For CNTF, addition of a third subunit, or α subunit, defines the high‐affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high‐affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high‐affinity LIF receptor, by adding a soluble form of the αCNTF receptor to the system to reconstitute the high‐affinity‐type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/αCNTF receptor bound to gp130 with an affinity of 3–5 × 10−10M, whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site comm

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041391.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one‐half, whereas NGF increased L1 protein levels ∼2.3‐fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10−7Mdexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down‐regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adre

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041400.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:To elucidate mechanisms regulating the production of platelet‐derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte‐enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum‐free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor‐β1(TGF‐β1) or tumor necrosis factor‐α (TNF‐α); the largest increase was detected after combined treatment with the two cytokines. Interleukin‐1β (IL‐1β) by itself had little or no effect but synergized with TGF‐β1in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A‐ and PDGF B‐chain mRNAs were detected in untreated astrocytes. PDGF B‐chain mRNA levels were increased by TGF‐β1, TNF‐α, TNF‐α/TGF‐β1, or IL‐1β/TGF‐β1, whereas PDGF A‐chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF‐β1, TNF‐α, and IL‐1β are able to stimulate astrocyte PDGF production. This cytokine network could play a ro

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041409.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We activated the death pathway in embryonic chick cerebral hemisphere neuron (E7CH) cultures with staurosporine (0.1–1.0 µM) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation (determined by Hoechst 33258 dye) associated with apoptosis.N‐Acylsphingosine (C2‐ceramide), a soluble ceramide analogue, was also able to induce apoptosis in these cells with the same characteristics and in the same time frame. We then observed that staurosporine was effective in inducing hydrolysis of sphingomyelin to ceramide as measured by a threefold increase in ceramide mass and increased incorporation of [3H]‐palmitate into ceramide, concurrent with activating the cell death program. Furthermore, the coaddition of a specific ceramidase inhibitor, oleoylethanolamine (15 µM), enhanced the formation of ceramide as well as the degree of DNA fragmentation and cell death. Exogenous addition of sphingomyelinase activated the death pathway whereas ceramide glycanase did not, and inhibitors of sphingomyelin or protein synthesis failed to block this type of killing. Our data suggest that the formation of ceramide from sphingomyelin is a key event in staurosporine‐induced and potentially all programmed

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.66041418.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY