摘要:

AbstractThe avian embryonic retina is widely used as a model system for cellular and molecular studies on central nervous system neurons. We aimed at the generation of cell lines from the early embryonic quail retina by retroviral oncogene transduction. For this, we made use of the retina organ culture system which exhibits both proliferation, necessary for stable oncogene transduction, and initial neuronal differentiation, a prerequisite for the generation of cell lines with mature neuronal properties. The oncogene myc was chosen ac it is both proliferation‐inducing and differentiation‐compatible. A chimeric gene, mycER, containing v‐myc and the hormone‐binding domain of the estrogen receptor, was used for transduction in order to allow for hormone regulation of myc activity. Transduced organ‐cultured cells from temporal and nasal retina were passaged into sparse single cell cultures. From these, colonies of rapidly dividing cells were isolated and the progeny expanded as cell lines. The lines contained cells with features of neuroepithelial cells, showing vimentin and A2B5. They also contained spontaneously differentiated neuronal cells showing neurofilament L and N‐CAM18O. A subpopulation of the neuronal cells exhibited the morphological characteristics of retinal ganglion cells, i.e., large pear‐shaped somata each emitting one long process with a distinct growth cone. In addition, they showed the marker profile of retinal ganglion cells, i.e., expression of Thy‐1, G4, DMGRASP, Nr‐CAM, neurofilament H, and tau. Neuronal differentiation could be induced by the addition of db cAMP and retinoic acid. The mature neuronal features of the lines open new possibilities to study properties of retinal neurons, including ganglion cells, in a defined and manipulable experimental system.

ISSN:0360-4012    

DOI:10.1002/jnr.490410402

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe modulation of acetylcholine‐activated current (IACh) by protein kinase C (PKC) was studied in Xenopus laevis oocytes microinjected with either mRNA extracted from C2C12 myotubes (C2C12 mRNA) or RNAs encoding murine αβγδ subunits of the nicotinic ACh receptor (nAChR). Voltage‐clamped oocytes were treated for 90 sec with 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA, 300 nM), a potent PKC activator. Transient increase in the amplitude and acceleration in the decay of IAChwere invariably observed within minutes of TPA application, and were independent of extracellular Ca2+concentration. Both parameters recovered to control within 20–30 min; then a slight depression of IAChdeveloped. By this time, an initial PKC down regulation was observed. At the peak of TPA‐induced potentiation, dose‐response relations suggested an increased binding affinity of nAChR for the neurotransmitter. 4a‐phorbol 12,13‐didecanoate (300 nM), a biologically inactive analogue of TPA, did not affect IACh, while staurosporine (5–10 μM), a potent inhibitor of PKC activity, suppressed the action of TPA on IACh. In oocytes co‐injected with C2C12 mRNA and with rat brain mRNA, IAChwas potentiated by 5‐hydroxytryptamine (10 μM), whose receptors are coupled to phosphoinositide hydrolysis. The nAChR‐channel activity in cell‐attached patches increased when TPA was applied to the oocytes. In 50% of the oocytes examined, a sustained depression of the single channel activity followed. We conclude that in Xenopus oocytes an endogenous PKC system regulates the function of embryonic‐typ

ISSN:0360-4012    

DOI:10.1002/jnr.490410403

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

年代:1995

数据来源: WILEY

摘要:

AbstractGlial fibrillary acidic protein (GFAP) and its mRNA, primarily expressed in astrocytes, are also expressed in peripheral nervous system Schwann cells as well as in certain non‐neural tissues. Schwann cells express a GFAP mRNA (GFAP‐β) which differs from the CNStype mRNA (GFAP‐β) by the presence of an extended 5′ untranslated region. We have developed a polymerase chain reaction assay which allows distinction of these two GFAP mRNAs, as well as quantitative analysis of their levels. In the cultured rat Schwannoma cell line RT4‐D6, GFAP‐b̃ was the major GFAP mRNA species, accounting for at least 75% of total GFAP (α+β) mRNA. GFAP‐b̃ was also detected in primary rat astrocyte cultures, where it constituted approximately 5% of the total GFAP mRNA, as well as in RNA samples prepared from normal rat cerebral cortex, and from hamster and human brain. In rat cortex, the temporal expression of GFAP‐β mRNA paralleled that of total GFAP mRNA, with plateau levels reached between postnatal days 15 and 20. In astrocyte cultures, the relative levels of GFAP‐α and ‐β mRNAs were differentially regulated by exposure to interferon‐γ (10 to 25 units/ml), which caused an increase in GFAP‐β levels while at the same time no change or a small decrease in total GFAP levels. In rat brain cortical slices, 4 hr exposure to 25 units/ml interferon‐γ decreased total GFAP mRNA levels over tenfold, while GFAP‐b̃ levels were unaffected. These data indicate that a second form of the GF AP mRNA is expressed in astrocytes both in vivo and in vitro and provide evidence for independent regulation of these two G

ISSN:0360-4012    

DOI:10.1002/jnr.490410404

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

年代:1995

数据来源: WILEY

摘要:

AbstractColchicine, nocodazol, and vinblastine, three microtubule‐disrupting drugs were shown to increase the levels of both nerve growth factor (NGF) mRNA and cell‐secreted NGF protein in L929 cells, with levels of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) or amyloid precursor protein (APP) mRNAs remaining unaffected. Northern blot analysis demonstrated that colchicine also increased NGF mRNA levels in rat primary astrocytes and mouse skin fibroblasts. The specificity of the effects observed was assessed by the fact that the microtubulestabilizing agent Taxotere®, a semisynthetic compound structurally related to taxol, suppressed the effects of colchicine, whereas lumicolchicine, a colchicine derivative that has no action on the microtubule network, had no influence on NGF expression. Likewise, the disruption of the microfilament network by cytochalasin B did not increase NGF mRNA levels in L929 cells. Furthermore, the increase in NGF gene expression observed following microtubule disruption depended on a cascade of events involving at least one protein kinase, which is not down‐regulated by phorbol ester, and on a pertussis toxin sensitive step. These results support the concept that tubulin and/or the microtubule cytoskeleton play an active role in the regulation of the NGF gene. © 1995 Wile

ISSN:0360-4012    

DOI:10.1002/jnr.490410405

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

年代:1995

数据来源: WILEY

摘要:

AbstractA decrease of corticotropin‐releasing factor (CRF) concentration has been reported in patients with Parkinson's disease (PD). The present study further examined the role of CRF in an animal model of parkinsonism induced by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Results indicated that both subchronic (2 days) and chronic (7 days) MPTP treatments decreased the number of CRF immunoreactive neurons in both the paraventricular nucleus (PVN) of the hypothalamus and the central nuelcus of the amygdala (ACN). This effect lasted for almost a month after withdrawal of chronic MPTP injections. In addition, nomifensine pretreatment protected against MPTP's toxicity on DA neurons, as assessed by tyrosine hydroxylase immunoreactivity in the substantia nigra. However, the same treatment did not prevent the toxicity of MPTP on CRF neurons. Further, no significant difference was notable in the number of CRF immunoreactive neurons between normal young adult and normal middle‐aged rats in both the PVN and the ACN. These results suggest that MPTP also produces a neurotoxicity on CRF neurons, and this effect is not secondary to MPTP's effect on DA neurons. Besides, altered CRF neuronal activity is involved in the process of pathological ageing, but not physiological ageing. Further, reduced CRF immunoreactivity in the PVN and ACN may imply alterations of neuroendocrine, autonomic as well as central functions caused by MPTP. © 1995 W

ISSN:0360-4012    

DOI:10.1002/jnr.490410406

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe describe an experimental model to study regeneration of lesioned corticospinal tract (CST) fibers in the adult rat spinal cord. After transection of all CST fibers at mid‐thoracic level the gap is grafted with a sterile, cell‐free collagen matrix. Two methods of collagen‐application are used: (1) injection of a fluid collagen solution into the lesioned area which self‐assembles in situ and (2) implantation of a solid collagen gel. At 4 weeks post‐implantation CST axons are anterogradely labelled with horseradish‐peroxidase (HRP). The collagen implant is evaluated for ingrowth of CST axons. The histopathological reaction (gliotic response) around the lesion and within the matrix is also studied. After application of a fluid collagen solution into the lesion area HRP‐labelled CST axons can be visualized within the implant. In addition, astroglial and reactive microglial cells invade the collagen‐matrix. On the other hand, if collagen is implanted as an already self‐assembled gel, no ingrowth of labelled CST axons nor of astroglial/reactive microglial cells is observed. Both methods of collagenapplication result in a considerable reduction of the gliotic response as compared to the ungrafted animals.We conclude that the method of application of collagen (i.e., fluid or gel) considerably affects the response of lesioned CST axons. The application of a fluid collagen graft which in situ self‐assembles is beneficial for the regrowth of lesioned CST axons in rat spinal cord. In this respect the formation ' of an astroglial scaffolding structure within the (fluid) collagen, probably due to optimal integration between host and graft, is very important. The inability of injured CST fibers to enter the solid collagen graft may be related to the absence of an astroglial scaffolding structure within the implant. ©

ISSN:0360-4012    

DOI:10.1002/jnr.490410407

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

年代:1995

数据来源: WILEY

摘要:

AbstractGM1, GD1a, and GT1b inhibit both PDGF‐stimulated and serum‐stimulated DNA synthesis in Swiss 3T3 cells and the human glioma cell line U‐1242 MG in a dose‐dependent manner. The ganglioside inhibitory effect is counteracted in a dose‐responsive fashion by serum such that ganglioside‐induced inhibition is essentially abolished in 10% serum. Because of the potentially important role that gangliosides play in growth regulation of human gliomas, this phenomenon was studied in detail using U‐1242 MG cells. Stimulation of DNA synthesis by low doses of serum in U‐1242 MG cells is inhibited in a dose‐responsive fashion by ganglioside GM1. However, serum itself counteracts the inhibitory effect of ganglioside in a dose responsive way. Kinetic analyses demonstrate that GM‐1 competes with some components of serum for sites on U‐1242 MG cells (Kb of GM1 =12.5 μM). On the other hand, GM1, GD1a, and GT1b stimulate DNA synthesis in quiescent U‐1242 MG cells in both sparse and confluent conditions, indicating that ganglioside‐stimulated DNA synthesis is dependent on the phase of cellular growth rather than cellular density. This growth stimulatory effect of gangliosides is more potent on quiescent, confluent cells than quiescent, sparse cells. These results demonstrate that exogenously added gangliosides can have opposite (bimodal) effects on progression of human glioma cells through the cell cycle depending upon the growth phase of the cel

ISSN:0360-4012    

DOI:10.1002/jnr.490410408

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe phenotypic expression of various neural cells is influenced by extracellular matrix (ECM) molecules. This study aims to develop a three‐dimensional gel tailored to support neurite extension from neural cells. Laminin‐derived (LN) oligopeptides CDP‐GYIGSR, a 19‐mer IKVAV containing sequence, GRGDSP, a cocktail of the three aforementioned LN peptides (PEPMIX), and a control peptide sequence GGGGG were covalently linked to an agarose hydrogel backbone using the bi‐functional coupling agent 1′1, carbonyldiimidazole. Embryonic day 9 chick DRGs and PC12 cells were suspended in three dimensions in underivatized and derivatized agarose gels and neurite extension was analyzed. Agarose gels derivatized with CDPGYIGSR and PEPMIX enhanced neurite outgrowth from DRGs while GRGDSP and IKVAV derivatized gels inhibited neurite extension when compared to underivatized agarose gels. The IKVAV derivatized gels significantly enhanced neurite outgrowth from PC12 cells in comparison to underivatized and other LN peptide derivatized gels. Agarose hydrogels carrying covalently immobilized LN oligopeptides thus evoke specific responses from cells which contain receptors to the peptides used. Agarose hydrogels derivatized with neurite promoting peptide sequences may find applications in various models of in vivo regeneration of nervous tissue. © 1995 Wil

ISSN:0360-4012    

DOI:10.1002/jnr.490410409

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

年代:1995

数据来源: WILEY

摘要:

AbstractRat astroglial cells were obtained either mechanically or by enzymatic dissociation and some properties of the primary and secondary cultures were studied. Differences in the ganglioside amount, taurine uptake, membrane fluorescence anisotropy, or their respective modulation by total brain gangliosides confirmed that although the two types of cultures are morphologically similar, they are biochemically distinct. © 1995 Wiley‐Liss, I

ISSN:0360-4012    

DOI:10.1002/jnr.490410410

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe have previously determined the presence of muscarinic receptors and the expression of several G proteins in homogenates and myelin fractions from ra sciatic nerves. In the present study we investigates whether changes in several signal transduction path ways in peripheral nerves might be responsible for some of the biochemical abnormalities (e.g., phosphoinositide metabolism) present in sciatic nerves from streptozotocin‐induced diabetic rats. Sciatic nerves from 5 week diabetic rats that were prelabelled with [3H]‐myo‐inositol displayed a significant increase in the basal release of inositol mono‐and bis‐phosphate, while carbamylcholine‐stimulated release was significantly smaller. Basal‐ and forskolinstimulated adenylyl cyclase activity was significantly decreased in sciatic nerve homogenates from diabetic animals. However, we were unable to detect any significant differences in the levels of cAMP in intact nerves or in nerve segments that were incubated in the presence or absence of forskolin. ADP‐ribosylation experiments showed that in sciatic nerves from experimentally diabetic rats there was a significant increase in the ADP‐ribosylation catalyzed by cholera and pertussis toxins. Measurements of the levels of a‐subunits of G proteins revealed that the expression of Gq/11α, Gsα, and Gi‐3α was increased by 30 to 50%. These results indicate that during the course of experimental diabetes, peripheral nerves exhibit an abnormal production of inositol phosphates and cAMP, together with an abnormal expression and/or function of G proteins. One of the consequences of such alterations is the diminished release of inositol phosphates triggered by muscarinic agonists in diabetic sciatic nerves

ISSN:0360-4012    

DOI:10.1002/jnr.490410411

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

年代:1995

数据来源: WILEY