摘要:

AbstractGrowth factors can induce both proliferation or differentiation of neuroblastoma (NB) cells through interaction with specific receptors. Using two automated colorimetric assays for determinations of cell numbers, the present study demonstrates that (a) different NB and neuroepithelioma cell lines show distinct responses, both qualitatively and quantitatively, to basic FGF (bFGF), NGF, and EGF(b) even closely related NB cell lines (e.g., SK‐N‐SH, SH‐SY5Y, and SHEP) do not respond uniformly to these factors; c) responses of the two neuroepithelioma cell lines employed (SK‐N‐MC and CHP‐100) differ, but match those of certain NB cell lines; and d) two growth factors, bFGF and EGF, may both stimulate or inhibit proliferation, depending on the cell line studied. Specifically, IMR‐32, SK‐N‐SH, and SH‐SY5Y showed a mitogenic response to each growth factor. Maximal proliferative responses ranged from 204–355% as compared to controls (100%). GICAN was stimulated by NGF (199%), and SK‐N‐MC and NMB by EGF (282 and 140%, respectively), but other factors were ineffective. CHP‐100 and GIMEN were inhibited by bFGF. NGF and EGF were not effective on CHP‐100 cells, while EGF caused an arrest of mitogenic activity in GIMEN cells, and NGF stimulated their proliferation. Cell lines SHEP and LAN1 did not respond to any factor. To begin to analyze putative relationships of growth factor responsiveness and growth factor/growth factor receptor expressions, IMR‐32, GIMEN, and LAN1 cell lines were studied for the presence of bFGF, NGF, FGF receptors (R)‐1 (flg) and FGFR‐4,trk, and low‐affinity NGF receptor (p75) mRNAs. All three cell lines expressed bFGF and NGF mRNA, but not the FGFR‐1, FGFR‐4,trk, and p75 mRNAs. These results suggest extremely diverse patterns of NB/neuroepithelioma cell responsiveness to “mitogenic” growth factors and no overt correlation between such responses and growth factor/growth facto

ISSN:0360-4012    

DOI:10.1002/jnr.490400602

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe HNK‐1 antibody recognizes a carbohydrate epitope expressed by many cell adhesion molecules in the nervous system that has been proposed to be an important adhesive determinant. This epitope is particularly prominent on the myelin‐associated glycoprotein (MAG) and is related to the antigenic target in an autoimmune mediated demyelinating neuropathy. Elucidation of the mechanisms underlying the biosynthesis and regulation of expression of the HNK‐1 epitope is therefore likely to have important functional and clinical implications. In order to investigate its biosynthesis and the regulation of its expression, we have expressed both human and rat MAG in several different cell lines by retroviral infection. These studies indicate that the cellular milieu determines whether the HNK‐1 epitope is expressed on the MAG polypeptide and provide an explanation for the significant variation in HNK‐1 levels that has been noted in different species. Using a transfected human neuroblastoma line, we have determined that this epitope is present on the fourth and/or fifth immunoglobulin‐like domain of rat MAG and that it is added intracellularly, probably in the trans Golgi. Finally we have found that expression of the HNK‐1 epitope is increased by activation of different second messenger systems, providing direct evidence that its expression can be regulated independently from that of the MAG polypeptide.© 1995 W

ISSN:0360-4012    

DOI:10.1002/jnr.490400603

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractRates of bovine photoreceptor gene transcription, as measured by nuclear run‐on assays, exhibit gene‐specific patterns of regulation. Here we investigate initiation and elongation in nuclear run‐on assays with the use of sarkosyl to further understand the nature of these gene‐specific elements. Opsin transcription, alone among several genes tested, proved sarkosyl‐sensitive. This sensitivity is maximal in adult retinas, with inhibition first detected in mid‐third trimester fetal retinas. Therefore, opsin transcription appears to involve different regulatory elements in adult and fetal retinas, implying a fetal to adult switch in the control of opsin gene expression. Although this regulatory switch is initially activated at a time when the fetal outer nuclear layer of the retina first achieves adult‐like morphology, further maturation of opsin regulation takes place postpartum since levels of sarkosyl sensitivity are almost 5‐fold greater in adult retinas compared to the 7.5 month fetus. We also show that the sarkosyl‐induced reduction of opsin transcripton is not due to prevention of de novo RNA polymerase II initiation in the run‐on reaction, suggesting the detergent alters a positive‐acting, postinitiation component of the transcriptional apparatus. Since levels of opsin transcription with sarkosyl are similar to those of the other visual transduction genes with or without sarkosyl, this detergent‐sensitive transcriptional component appears to account for the singularly high, gene‐specific rate of opsin transcription in retinal photoreceptor cell

ISSN:0360-4012    

DOI:10.1002/jnr.490400604

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractGrowth factors differently regulate astroglial cell differentiation and proliferation. In an effort to understand the early intracellular events promoted by growth factors in astroglial cells, we have determined the effects of insulin‐like growth factor I (IGF1), insulin, platelet‐derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factors (FGFs) on phosphatidylinositol‐3 kinase (PI(3)‐kinase).In astroglial cells cultured in serum‐free medium, IGF1, PDGF, and EGF, which stimulate cell proliferation, increased PI(3)‐kinase activity immunoprecipitated with anti‐phosphotyrosine antibodies as shown by thin layer chromatography and high performance liquid chromatography. FGFa and FGFb, which strongly stimulate proliferation, glutamine synthetase, and deiodinase activities and modify cell morphology, have no effect on PI(3)‐kinase activity. Addition of 1 nM PDGF, 10 nM IGF1, or 100 nM EGF to the culture medium rapidly stimulated PI(3)‐kinase activity which declined slowly after 2 min. The stimulation of PI(3)‐kinase increased with growth factor concentration. The maximum increase in PI(3)‐kinase activity occurred with 50 nM IGF1, 1 nM PDGF, or 100 nM EGF. Since insulin was active only at high concentration (1 μM), its effect was probably mediated through IGF1 receptors and not through IGF1 receptors and not through insulin receptors.IGF1 and PDGF, to a lesser degree, also increased the PI(3)‐kinase activity associated with pp60c‐srcprotein. Immunoblots performed with an antibody directed against the p85‐subunit of the PI(3)‐kinase confirmed that IGF1 increased the number of PI(3)‐kinase molecules associated with phosphotyrosine‐containing proteins or with c‐src protein.Each growth factor affects in a different manner the association of PI(3)‐kinase with phosphotyrosine‐containing proteins and with pp60c‐srcand thus probably modulates intracellular signals downstream of PI(3)‐kinase

ISSN:0360-4012    

DOI:10.1002/jnr.490400605

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractAn in vitro slice preparation of rat amygdala was used to study the actions of forskolin and cyclic adenosine‐3′,5′‐monophosphate (cAMP) analogues on the N‐methyl‐D‐aspartate (NMDA) receptor‐mediated synaptic potential (EPSPNMDA). Intracellular recordings were made from basolateral amygdala neurons in the presence of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10μM) and picrotoxin (50 μM) to pharmacologically isolate the EPSPNMDA. Application of forskolin (25 μM) markedly and persistently potentiated the EPSPNMDAIn contrast, the inactive forskolin analogue, 1,9‐dideoxy‐forskolin, failed to affect the EPSPNMDAsignificantly. Superfusion of dibutyryl‐cAMP (dbcAMP, 200 μM) for 15 min caused a transient depression of the amplitude of EPSPNMDA. The EPSPNMDAamplitude was reduced to 68 ± 3% of control (n = 10) 15 min after the application, restored to its control value within 25 min, and followed by a long‐term potentiation (LTP). Pretreating the slices with 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 5 μM), a selective A1receptor antagonist, blocked the transient depressive phase produced by dbcAMP. This result suggests that the transient depression induced by dbcAMP was likely due to the interaction of dbcAMP or its breakdown products with adenosine A1receptors.To determine the site of action, we examined the effect of forskolin on the postsynaptic responses to exogenously applied NMDA. Forskolin potentiated the postsynaptic depolarization induced by NMDA, suggesting that the enhancement is mediated, at least in part, by a persistent upregulation of postsynaptic NMDA receptor‐operated conductances.Occlusion experiments were performed to examine whether the sustained enhancements of EPSPNMDAproduced by tetanic stimulation (TS) and forskolin share a common mechanism. Three episodes of TS were delivered to saturate the LTP and, under this condition, forskolin still caused a further potentiation of the EPSPNMDA. Similarly, TS, delivered after the EPSPNMDAwas enhanced by forskolin or dbcAMP, produced LTP. These results suggest that the long‐term enhancements of EPSPNMDAproduced by TS and forskolin are different and thus do not support the hypothesis that activation of prot

ISSN:0360-4012    

DOI:10.1002/jnr.490400606

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractMacrophage inflammatory protein 1 (MIP‐1) is a recently characterized inflammatory and chemokinetic cytokine. Proinflammatory stimuli have been shown to induce expression of MIP‐1 by macrophages. We hypothesized that microglia and astrocytes express MIP‐1α because of their many immunologic similarities to macrophages. MIP‐1α mRNA was examined with quantitative reverse transcription and polymerase chain reaction in an immortalized mouse microglial cell line (BV‐2) and in mouse cortical astrocyte cultures. We found that in both the BV‐2 microglial cell line and in astrocyte cultures, MIP‐1α mRNA was strongly induced by lipopolysaccharide and the phorbol ester PMA. MIP‐1α mRNA was reduced by dBcAMP, interferon‐γ, and PGE1. Dexamethasone decreased MIP‐1α mRNA levels in astrocyte cultures, but not in BV‐2 microglial cells. Interleukin‐1β, tumor necrosis factor α and MIP‐1α had no effect on MIP‐1α mRNA is expression. These findings demonstrate that MIP‐1α mRNA is expressed by cultured glial cells and is regulated by proinflammatory and anti‐inflammatory stimuli. MIP‐1α may be expressed by microglia and astrocytes in vivo, and may help modulate

ISSN:0360-4012    

DOI:10.1002/jnr.490400607

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractTo study the sequence of degenerative events possibly associated with cholinergic cell death in Alzheimer's disease, septal cholinergic neurons derived from rat embryonic brains were exposed to chronic excitotoxic stress by glutamate. Counts of choline acetyltransferase (ChAT)‐immunopositive neurons and measurement of ChAT activity revealed that concentrations of glutamate on the order of 70 μM killed 50% of cholinergic neurons after 24 hr of treatment. Neurotoxic effects were not aimed at cholinergic neurons specifically, since other populations of cells present in these cultures were also affected at similar concentrations. The noncompetitiveN‐methyl‐D‐aspartate (NMDA) receptor channel antagonist MK‐801 (10 μM) abolished acute neuronal swelling and rescued from late degeneration both cholinergic and non‐cholinergic cells when concentrations of glutamate up to 500 μM were added to the cultures. Protective effects declined progressively with increasing concentrations of the amino acid, even when MK‐801 was raised to its highest nontoxic levels, e.g., 50 μM. The kainate/quisqualate receptor antagonist CNQX provided no protection alone or in combination with MK‐801. Nerve growth factor (NGF), used in standard culture conditions to stimulate the expression of the cholinergic phenotype, was shown not to influence excitotoxic neurodegenerative changes. Several observations suggested that nitric oxide (NO) may act as an intercellular messenger of NMDA‐mediated cell death in septal cultures: (1) Most of the cholinergic neurons contained the NO synthase enzyme as characterized by NADPH‐diaphorase (NADPH‐d) staining; (2) sodium nitroprusside (SNP) [a chemical with the ability of generating NO] was capable of mimicking some of the aspects of the glutamate‐induced degenerative process; (3) the rise in cyclic GMP which was observed in the presence of toxic levels of glutamate and which is usually taken as an index of NO production, was antagonized by MK‐801 and by the inhibitor of the NO synthase enzyme, L‐NOARG. Yet, the fact that L‐NOARG and its congener, L‐NAME, were ineffective in preventing glutamate‐induced neurodegenerative changes in our culture system did not substantiate our working hypothesis. Altogether these results suggest that glutamate‐induced cholinergic neuronal death is the consequence of an overstimulation of NMDA receptors and that neither NGF nor NO plays a key role in the degen

ISSN:0360-4012    

DOI:10.1002/jnr.490400608

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractA mouse monoclonal antibody (5B9), directed against a carbohydrate epitope of human epidermal growth factor receptor (EGFR), recognized an 81‐kDalton glycoprotein in buffer‐soluble and detergent‐solubilized rat brain extracts (BE). The glycoprotein was more abundant in extracts prepared from injured brain than in those from normal tissue. Removal from BE of the antigens recognized by 5B9 increased their astrocyte mitogenic activity. Sections of injured rat brain and cultures derived from damaged brain, enriched in microglia, showed 5B9 immunoreactivity in ED1‐positive cells. The abundance of the glycoprotein recognized by 5B9 in injured, relative to normal, tissue, suggested that molecules with EGFR immunoreactivity may be expressed in reactive microglial cells and released after injury. © 1995 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490400609

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe defective parts of the visual field of two braininjured patients were stimulated with different spots of light. There is evidence for at least five independent visual functions which can be restored due to constant stimulation of the blind part of the visual field: (1) The constant stimulation of the blind part of the visual field with spots of white light leads to an increase of the visual field for the perception of white light only. (2) The constant stimulation with spots of light of different wavelengths leads to an increase of the visual field for different color perception. To enlarge the visual field for the perception of the color red, a light stimulus with the wavelength of 656 nanometers (nm) was used; for the visual field for the perception of the color green 525 nm; for yellow 578 nm; and for blue 450 nm. (3) The constant stimulation of the blind visual field with black and white light bars of different orientations and constellations leads to an increase of the foveal acuity and an improvement of form perception in the periphery of the visual field.The results suggest that the recovery of visual functions, different color perception and form perception, may depend upon neuronal regeneration in the human visual cortex; regeneration occurs with adequate and constant stimulation of its specific neurons. © 1995 Wiley‐Liss, I

ISSN:0360-4012    

DOI:10.1002/jnr.490400610

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractOne of the endogenous substances which modulate glutamate receptor binding was isolated and highly purified from porcine brain. The purification involved extraction of brain tissue with doubled distilled water, followed by gel filtration, anion exchange, cation exchange, and several steps of C18 reverse‐phase high performance liquid chromatography (HPLC). A low molecular weight glutamate binding inhibitor (LGBI) was purified to apparent homogeneity as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at 220 nm. The LGBI appears to be a small molecule (<2 kD) that is heat‐and acid/base‐stable. The highly purified LGBI has no effect on GABAAand benzodiazepine receptor binding. The LGBI is not L‐glutamate, L‐aspartate or other negatively charged endogenous substances, since they are clearly separated from the LGBI in anion exchange chromatography. The inhibitory effect of the LGBI on [3H]L‐glutamate binding is reversible, and it only changes the Bmaxwhile the Kdremains the same. Since the membrane preparations used for [3H]L‐glutamate binding assays for the detection of LGBI activity were enriched with quisqualate (QA)‐sensitive subtypes, it was suggested that the LGBI could be a modulator of the QA receptor. Some amino acids which produce significant inhibition of glutamate binding activity were also compared with the LGBI, and they all showed no resemblance to the LGBI. The chemical structure of the LGBI remains to be determined. © 1995

ISSN:0360-4012    

DOI:10.1002/jnr.490400611

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY