摘要:

AbstractTumor necrosis factor‐α (TNF‐α), a proinflammatory cytokine, has long been known to be involved in the pathogenesis of central nervous system infections and of certain neurodegenerative diseases. However, the possible role of the blood‐brain barrier (BBB), the active interface between the blood circulation and brain tissue, remained unknown during these pathological conditions. In our in vitro reconstructed BBB model, 1‐hr exposure of recombinant human TNF‐α (in concentrations of 50, 250, and 500 U/ml, respectively) to the luminal membrane of bovine brain capillary endothelial cells (BBCEC) did not change significantly the transendothelia: flux of either sucrose (m.w. 342 Da), or inulin (m.w. 5 kDa) up to 4 hr (early phase), except for a slight decrease (P<0.05) in sucrose permeation at 2–4 hr with the highest dose of TNF‐α On the other hand, at 16 hr after the 1‐hr challenge with TNF‐α (delayed phase) at all 3 concentrations, significant increase was induced in the permeability of BBCEC monolayers for both markers. These changes of permeability were accompanied by a selective reorganization of F‐actin filaments into stress fibers, while the intracellular distribution of vimentin remained similar to the control. These results suggest that BBCEC can respond directly to TNF‐α by a delayed increase of permeability and reorganization of actin filam

ISSN:0360-4012    

DOI:10.1002/jnr.490410602

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe glucocorticoid hydrocortisone (HC), applied for up to 2 weeks to either aneurally or innervated cultured human muscle, produced 2‐fold increase of the number of dihydropyridine ([3H]PN200‐110) binding sites. The K+‐induced, nifedipine‐inhibited Ca2+uptake was increased 40%. The effect of HC was concentration‐ and time‐dependent. [3H]PN200‐110 affinity for its receptor was not affected by HC treatment. HC did not exert significant influence on the total amount of protein, CK activity, and the number of myotubes. These results indicate that voltage‐dependent L‐type Ca2+channel expression in human muscle is regulated by glucocorticoid. © 19

ISSN:0360-4012    

DOI:10.1002/jnr.490410603

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe found previously that epidermal growth factor (EGF) decreases choline acetyltransferase (ChAT) activity in forebrain cholinergic neurons in vitro indirectly via glia (Kenigsberg et al.: Neuroscience 50:85–97, 1992). However, which glial type(s) are implicated in this response remained to be determined. Here we report that in primary cultures from the fetal rat medial septal area the complete elimination of oligodendrocytes or partial elimination of microglia from these cultures does not change the cholinergic cell response to EGF. However, the elimination of astroglia in our cultures by α‐aminoadipic acid treatment blocks EGF's effects on the chofinergic neurons. Co‐culture experiments using pure neuronal and purified glial cells from the medial septum further demonstrate that the cholinergic cell response to EGF can be maintained in the presence of astroglia only. In addition, it appears that EGF regulates the release of soluble factors from pure astroglial cultures following their peak mitotic response to EGF that decreases ChAT enzymatic activity. This soluble cholinergic neuromodulatory activity found in conditioned media from EGF‐treated astrocytes has a molecular weight greater than or equal to 10 kD and loses potency following multiple freeze‐thaw cycles.These results suggest that a direct glial cell response to a specific glial growth factor like EGF may have an important impact on the expression of local neurons, like the cholinergic in the forebrain. © 1995 Wile

ISSN:0360-4012    

DOI:10.1002/jnr.490410604

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

年代:1995

数据来源: WILEY

摘要:

AbstractExpression of acetylcholine receptor and of the asymmetric molecular forms of acetylcholinesterase (AChE) in the extrajunctional regions of rat muscles is suppressed during early postnatal development. In mature muscles, the extrajunctional synthesis of acetylcholine receptor, but not of the asymmetric molecular forms of AChE, becomes reactivated after denervation. The hypothesis that a denervated muscle needs reinnervation in order to revert transiently to an immature state characterized by high extrajunctional production of the asymmetric AChE forms, was examined in rat muscles recovering after nerve crush. Molecular forms of AChE were analysed by velocity sedimentation. Activity of the asymmetric A12 AChE form in the extrajunctional regions of the slow soleus (SOL) muscle increased during the first week after reinnervation to about 9 times its control level, remained high for about one week, and declined towards normal thereafter. If the nerve was crushed close to the muscle and reinnervation occured very rapidly, the extrajunctional increase of the A12AChE form still occured but was less pronounced than after late reinnervation. In contrast, a transient paralysis of the SOL muscle due to acetylcholine receptor blockade by α‐bungarotoxin, followed by spontaneous recovery of muscle activity after 3–5 days, did not revert AChE regulation into an immature state. Disuse of the SOL muscle caused by leg immobilization, which is known to change the tonic pattern of neural stimulation of the SOL muscle into a phasic one, did not prevent the reversion of AChE regulation during reinnervation. This indicates that neural stimulation pattern is not crucial for this reversion. In contrast to slow SOL, the fast extensor digitorum longus muscle did not revert to an immature state in respect to AChE regulation after reinnervation. This muscle type‐specific response may be due to intrinsic differences between the myogenic cells of slow and fast muscle fibres. © 1995 Wiley‐

ISSN:0360-4012    

DOI:10.1002/jnr.490410605

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe role of extracellular nucleotides in intracellular signalling and neurosecretion was assessed in PC12 cells. Activation of phospholipase C and increased [Ca2+]iwere mediated by purinoceptors with an agonist potency profile, ATP ∼ UTP>2‐methylthioadenosine triphosphate (2‐MeSATP), typical of P2U. ATP also evoked a rapid acidification followed by a more gradual alkalinization (measured with 2′,7′‐biscarboxyethyl‐5(6)‐carboxyfluorescein (BCECF)), while UTP induced only a gradual alkalinization. The amiloride analogue 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA) attenuated the alkalinization phase suggesting activation of the Na+/H+exchanger by ATP and UTP. Using bisoxonol and [3H]tetraphenylphosphonium ([3H]TPP+) as potential‐sensitive probes, we showed that while ATP rapidly depolarized PC 12 cells in an Na+‐dependent manner, UTP evoked a much reduced and delayed response. The potency profile (ATP ∼ 2‐MeSATP ∼ adenosine 5′‐0‐(3thiotriphosphate) (ATPγS) ≫ UTP, α, beta;‐methyleneATP) suggested involvement of a receptor subtype distinct from P2U. Secretion of endogenous dopamine was also assessed. Those nucleotides that induced depolarization (ATP, 2‐MeSATP, ATPγS) were also the most potent secretagogues. UTP was ineffective. Our results suggest that ATP stimulates distinct purinoceptor subtypes and induces neurosecretion through the activation of mult

ISSN:0360-4012    

DOI:10.1002/jnr.490410606

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

年代:1995

数据来源: WILEY

摘要:

AbstractImmortalized neuroectodermal precursor cell lines were generated from mouse brain by the SV40 large T antigen expressed under the control of the LAP (lac activating protein) mammalian regulatory system. The LAP system permits the reversible expression of T antigen as a function of the exogenous inducer, isopropyl‐β‐D‐thiogalactopyranoside. Immortalized cells can be stably maintained in an undifferentiated state in monolayer cultures. Cell lines expressed the early neurofilament‐like protein nestin, but not markers characteristic for mature cells such as the neurofilament light protein and glial fibrillary acidic protein. Downregulating the LAP‐controlled T antigen with isopropyl‐β‐D‐thiogalactopyranoside was not sufficient to induce differentiation. However, when cells formed three‐dimensional aggregates, differentiation to a neuronal phenotype occurred, indicating that cell‐cell interaction plays an important role in their differentiation. Cells in aggregates did not proliferate, even in the presence of T antigen, suggesting that an aggregation‐induced signal to cease growth was dominant over the growth signal of T antigen. Further morphological differentiation was induced by basic fibroblast growth factor. These immortalized cells should facilitate molecular and cellular studies concerned with the mechanism of commitment, fate determination, and mitotic arrest of neuronal precursor cells in the developing mammalian CNS.

ISSN:0360-4012    

DOI:10.1002/jnr.490410607

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

年代:1995

数据来源: WILEY

摘要:

AbstractWe used molecular modeling techniques to examine six reported antagonists of glycine with varying K; values against strychnine. We found the data suggest two groups operating with different mechanisms. In group 1 (strychnine, brucine, Pitrazepin, and bicuculline methobromide) the antagonist contains two or three sites that can electrostatically bind to the three comparable groups of opposite charge in the recognition site where the natural neurotransmitter binds, thus opening the chloride channel. In addition, when in this position, the antagonist is able to also block the now opened chloride channel with a different portion of its structure. In many cases, this involves an interaction between a carbonyl group, on the antagonist and the guanidinium group of arginine which is part of the polypeptide segment of the outer mouth of the chloride channel (Grenningloh et al., Nature 330:2526, 1987). In group 2 (R5135 and 1,5‐diphenyl‐3,7‐diazaadamantan‐9‐ol) the antagonist contains charged sites but when one of these molecules attaches to the recognition site, the chloride channel is not opened. In addition, R5135 contains a carbonyl group which attaches to arginine as pointed out in the text, whereas 1,5‐diphenyl‐3,7‐diazaadamantan‐9‐ol contains a phenyl group that can block the channel. ©

ISSN:0360-4012    

DOI:10.1002/jnr.490410608

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

年代:1995

数据来源: WILEY

摘要:

AbstractSH‐SY5Y cells differentiate into neuronal‐like cells and express marker proteins like growth‐associated protein (GAP‐43) and neuropeptide tyrosine when treated with a low concentration (16 nM) of the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in the presence of growth factors or serum. Both control and differentiated cells expressed protein kinase C‐α (PKC‐α), PKC‐ε, and PKC‐ζ as revealed by Western blot analyses, but the subcellular distribution of the three isoforms was not uniform, indicating specific localized functions of the enzymes. In growth cones prepared from differentiating cells PKC‐α and PKC‐ε were enriched. In contrast, PKC‐ζ was more evenly distributed within the differentiating cell. Cells treated with a high concentration of TPA (1.6 μM) differentiate poorly and continue to proliferate. In those cells, PKC‐α and PKC‐ε were found to be down‐regulated while PKC‐ζ remained present. Thus, down‐regulation of PKC‐α and PKC‐ζ appears to be incompatible with neuronal differentiation of SH‐SY5Y cells. These cells also differentiate when treated with a combination of basic fibroblast growth factor and insulin‐like growth factor I. Growth cones isolated from such cells are also enriched in PKC‐α and PKC‐ε, but not in PKC‐ζ. Based on the subcellular distribution of PKC‐α and ε, and that PKC substrates like GAP‐43 and pp60c‐srcare enriched in SH‐SY5Y

ISSN:0360-4012    

DOI:10.1002/jnr.490410609

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe P19 embryonal carcinoma cell line is a useful model system for analyzing the factors that regulate neuronal differentiation. In order to analyze the extrinsic factors that are involved in differentiation, it is necessary to carry out experiments in fully defined media. Here we have investigated the neuronal differentiation of P19 cells in two defined media. Cells that are propagated and induced with retinoic acid in standard serum‐containing medium are capable of differentiating into neuron‐like cells in N2 medium. Dividing fibroblast‐like cells also appeared in these cultures. After about 10 days in culture in N2 medium, the great majority of neuron‐like cells died. On the other hand, culturing induced cells in N2 medium for 5 days and then switching to a defined medium consisting of Neurobasal medium plus B27 supplement allowed the neuron‐like cells to survive for prolonged periods of time. This defined medium thus provides a suitable system for analyzing extrinsic factors that affect the survival and differentiation of P19 neurons. P19 cells induced with retinoic acid and plated in N2 were exposed to bFGF and EGF, which are known to be mitogens for neuronal precursor cells. Both growth factors were mitogenic for a subpopulation of the induced cells. In separate experiments, cells cultured in N2 in the presence of RA were induced to differentiate into neuron‐like cells. © 1995 Wil

ISSN:0360-4012    

DOI:10.1002/jnr.490410610

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

年代:1995

数据来源: WILEY

摘要:

AbstractHSP27 expression was investigated in cultured neurons and glial cells isolated from fetal human brains using immunoblotting and immunocytochemistry. Under unstressed conditions, HSP27 was identified at a high level in astrocytes (>99%), at a low level in neurons (7%), and at a minimally detectable level in microglia (<1%), whereas it was undetectable in oligodendrocytes. Under these conditions, HSP27 was located in the cytoplasm, fractionated into the Triton X‐100‐soluble phase, and composed chiefly of the basic isoform (HSP27a). After exposure to heat stress (43°C90 min), the level of HSP27 exproion ryas not altered in astrocytes but was elevated significantly in neurons (11–21%) and microglia (4–7%) during 8–48 hr postrecovery periods, while it remained undetectable in oligodendrocytes. In addition, various human neural cell lines exhibited differential patterns of HSP27 expression under unstressed and heat‐stressed conditions. Following heat shock treatment (45°C/30 min), granular aggregates of HSP27 were identified in the cytoplasm of astrocytes. Under heat‐stressed conditions, HSP27 was distributed within the Triton X‐100‐insoluble fraction associated with an increase in two more acidic isoforms (HSP27b and HSP27c). HSP27 and αβ‐crystallin were coexpressed in astrocytes under unstressed and heat‐stressed conditions. When astrocytes were exposed to known HSP27 inducers, hydrogen peroxide and cysteamine reduced the synthesis of HSP27, while estradiol showed no effects. The differential patterns of constitutive and heat‐induced expression of HSP27 in cultured human neurons and glial cells suggest that the cellular mechanisms by which HSP27 expression is regulated are different among various cell types in the human central nervous sys

ISSN:0360-4012    

DOI:10.1002/jnr.490410611

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

年代:1995

数据来源: WILEY