摘要:

AbstractWe have determined the cellular distribution of calbindin D28KmRNAs throughout the mouse brain by in situ hybridization. While these studies identified neuronal populations similar to those previously identified in rat brain by immunohistochemistry, some discrepanicies exist. These may derive from species differences or from the immunological cross‐reactivity of calbindin D28Kantiserum with other proteins. We note an intriguing association between the distribution of neurons containing calbindin D28KmRNA and those reported by others to contain the inositol 1,4,5‐triphosphate (InsP3) receptor. © 1994 Wiley‐Lis

ISSN:0360-4012    

DOI:10.1002/jnr.490370302

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe human neuroblastoma cell line, SH‐SY5Y, differentiates into a neuronal, sympathetic phenotype in the presence of phorbol ester and serum. Growth cones prepared from differentiating SH‐SY5Y cells have characteristics similar to those of growth cones from embryonic rat brain. In addition, SH‐SY5Y growth cones contain ribosomes. In this study we show, by metabolic labeling of isolated growth cones, that local protein syntheisis occurred in these structures. The pattern of labeled proteins was very similar to that of the corresponding cell body fraction. RNA was shown to be transported to the growth cone compartment, and by in situ hybridization. β‐actin mRNA could be visualized in intact neuritic growth cones. Comparison by Northern blot hybridizations of RNA prepared from growth cones and cell bodies, respectively, showed that mRNAs coding for growth‐associated protein 43, microtubule‐associated protein 2, actin, neuropeptide tyrosine, and glyceraldehyde‐3‐phosphate dehydrogenase were present in both fractions. In contrast, mRNAs coding for the nuclear proteins c‐junand N‐mycwere virtually absent in the growth cone, but readily detectable in the cell body preparation. The selective distribution of mRNAs to the growth cones was not restricted to stable, abundant mRNA species, since mRNA coding for the insulin‐like growth factor I receptor was stable, but not present in growth cones. Thus, differentiating SH‐SY5Y cells can sort and transport RNA to the growth cone compartment, suggesting that this system of clonal cells could be useful to unravel mechanisms involved in the compartmentalization of mRNA

ISSN:0360-4012    

DOI:10.1002/jnr.490370303

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

年代:1994

数据来源: WILEY

摘要:

AbstractIt is generally accepted that the action of thyroid hormones is mediated through specific nuclear receptors. Recent studies have demonstrated the homology of the thyroid receptor with the cellular product of the oncogen v‐erbA. So far, two genes have been identified and classified as α and β subtypes. In this study, the expression of nuclear triiodothyronine (T3) receptors (NT3RS) was examined in secondary cultures containing 85–90% oligodendrocytes (OL) prepared from newborn rat brain primary cultures enriched in OL. These cultures, which are able to produce myelin membranes, were examined by double immunolabelling with a monoclonal antibody (2B3) raised against purified rat liver NT3Rs and with antibodies against two maturation markers of OL: an early marker, galactocerebroside (GC), and myelin basic protein (MBP), which is expressed later than GC. 2B3 recognized three nuclear proteins with the same molecular weights as β, α1, and α2 sybtypes with different capacities for binding T3. In 5‐day ‐old OL secondary cultures (25 days, total time in culture), 2B3‐NT3R immunoreactivity was located in 77% of morphologically immature OL (GC)+cells, whereas only 44% of morphologically mature OL were immunoreactive. Only 35% of the MBP+cells co‐expressed NT3Rs. In the corpus callosum of devloping rat brain, at all ages studied from 7–60 days postnatal, the total absence of NT3Rs in dark OL (morphologically mature), confirmed by ultrastructural immunocytochemistry, indicates an even more dramatic decrease during maturation. Furthermore, the percentage of medium OL (less mature) stained by 2B3 is reduced by approximately half in 60‐ compared to 20‐day‐old rat brain. It is of interest to note that the in vitro observation with maturation markers mirrors the in vivo decrease of NT3R expression during development. It is intersting that NT3Rs are absent in vivo before the critical period of active myelination. These data indicate the presence of a nuclear T3binding protein in the nuclei of OL at the time of myelination both in vitro and in vivo. The transient expression of these NT3Rs during active myelination argues in favour of a direct effect of thyroid hormones on OL

ISSN:0360-4012    

DOI:10.1002/jnr.490370304

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe expression of the neural cell adhesion molecule (N‐CAM) and α 2–8 linked polysialic acid (PSA), which is believed to be predominantly expressed on N‐CAM, was investigated during early embryonic development of the mouse (embryonic days 7.5 to 10.0). By immunocytochemistry, in tissue sections, N‐CAM and PSA were not detectable at embryonic day 7.5 but were expressed in the prominent body regions such as somites, unsegmented mesoderm, developing heart, and neuroectoderm at embryonic day 8.0 N‐CAM and PSA immunoreactivities were always predominantly associated with the plasma membrane. No tissue could be detected which was positive for PSA but negative for N‐CAM. In Western blot analysis of whole embryos, by contrast, only the lightly sialylated and PSA‐negative 180 and 140 kD isoforms of N‐CAM were present at embryonic day 8.0 and strong expression of PSA‐bearing, heavily sialylated N‐CAM was not detectable before embryonic day 10.0. In Western blot analysis of N‐CAM immunoaffinity purified from whole embryos and digested with neuraminidase as well as in Northern blot analysis, the 120 kD isoform of N‐CAM or its corresponding mRNA were not expressed in detectable amounts during the time period investigated

ISSN:0360-4012    

DOI:10.1002/jnr.490370305

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe extracellular matrix glycoprotein tenascin is expressed in the developing mouse cerebellum as a group of four protein species of different molecular weights. The difference is most likely due to alternative splicing which is known to occurr in tenascin mRNA isoforms that would account for this heterogeneity, tenascin splice variants were isolated from mouse brain by the polymerase chain reaction (PCR). In agreement with Northern blot analysis, amplification by PCR revealed a general decrease in tenascin mRNA expression during development from embryonic and early postnatal to adult stages. This decrease was more pronounced for isoforms of high molecular weight compared to those of low molecular weight. In accord with the observations at the protein level, four splice variants were found to be predominantly expressed, containing insertions of either six, five, or one fibronection type III repeat, or comprising no insertion. In addition, a minor splice variant with an insertion of four fibronectin type III repeats was isolated. Three of the isolated mRNA splice variants have not yet been described for mouse tenascin. Among them, an isoform containing six alternatively spliced repeats was found to include a novel fibronectin type III repeat. The sequence of this repeat displays 96.7% similarity to a corresponding type III repeat in human tenascin, revealing a strict evolutionary conservation between tenascin molecules from different species in the region of alternative splicing. Southern blot analysis of the amplified mRNA isoforms showed that the novel mouse type III repeat is confined to splice variants with an insertion of six fibronectin type III repeats. Furthermore, in situ hybridization on sections from mouse embryos indicated that tenascin‐specific mRNAs containing the novel type III repeat are predominantly expressed in the central nervous system. © 1994 Wiley‐Liss,

ISSN:0360-4012    

DOI:10.1002/jnr.490370306

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe amyloid precursor protein (APP) is widely distributed within the CNS, where it is expressed in both neurons and glia. We have isolated axolemma and periaxolemmal‐myelin from rat brain and have determined by Western blot that APPs, Mr 100–110 kDa, are major constituents of these membrane. Isolation of axolemma, periaxolemmal‐myelin, and compact myelin show that while APP represents 1 and 0.6% of the proteins of these respective membranes, it is absent from compact myelin. These results indicate that APP transported down the axon is deposited at sites in the axolemma as well as the synapse, and that within the myelin complex, APP is targeted to the periaxolemmal domain. Both axolemma and periaxolemmal‐myelin contained a 10.5 kDa APP peptide which, based on reactivity with anti‐C‐terminal APP antibodies but not with anti‐N‐terminal antibody, appears to be a membrane‐associated C‐terminal fragment. Western blots with antibodies to Alzheimer precursor‐like proteins (APLP) indicate that APP immune reactivity is not a result of cross reactivity with APLPs. Isolation of axolemma from human autopsy material showed nearly identical results with a clear enrichment, relative to homogenate, of APP Mr 100–100 and the 10.5 kDa C‐terminal peptide. The demonstration of APP in axolemma and periaxolemmal‐myelin was replicated in membrane isolated from bovine brain. Bovine studies were extended to analysis of white matter clathrin‐coated vesicles; these data show that coated vesicles isolated from white matter, under conditions that previous studies indicate are largely endocytic vesicles, contain levels of APP comparable to that found in axolemma and periaxolemmal‐myelin. In addition, these vesicles contain cysteinly and aspartyl proteases. Incubation of axolemma with cathepsin B at pH 6.0 caused a rapid loss in the immune reactivity of APP Mr 100–110 and Mr 10.5 when analyzed with antibodies to APP672–695. This appears to be the result of hydrolysis within the epitope and not proteolysis of APP or the C‐terminal peptide, since no loss of reactivity was observed when analyzed with antibodies to sites more distal to the C‐terminus. Thus, cathepsin B hydrolyses membrane bound APP close to the C‐terminus and may be a useful tool for altering C‐te

ISSN:0360-4012    

DOI:10.1002/jnr.490370307

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

年代:1994

数据来源: WILEY

摘要:

AbstractMost fetal rat brain cells expressing the embryonal, highly sialyated form of the cell adhesion molecule N‐CAM (E‐N‐CAM) are precursor cells, as jugded from the absence of marker molecules specific for mature neural cell types. However, the detection of E‐N‐CAM+cells in frozen sections does not provide information on the lineage‐specific differentiation of these cells during development. To investigate their differentiation behaviour in vitro, E‐N‐CAM+cells were isolated at different times of brain development by fluorescence‐activated cell sorting (FACS), using a monoclonal antibody (Mab RB21‐7) which specifically recognizes polysialic acid (PSA) residues on E‐N‐CAM. Double‐immunofluorescence analyses showed that ht emajority of E‐N‐CAM+cells isolated on prenatal days 15 to 18 differentiated into neurons while a small subset of Mab RB21‐7 binding cells proved to be astrocytic precursors and/or bipotential. The proportion of E‐N‐CAM+astrocytic precursors increased during later development (prenatal day 22) concomitantly with the onset of gliogenesis. While conversion of E‐N‐CAM to mature forms of N‐CAM was never observed in neurons during cultivation, E‐N‐CAM+cells of the astrocyte lineage switched to N‐CAM soon after the onset of GFAP expression. A lineage‐specific transition of E‐N‐CAM to mature N‐CAM expression is, therefore, suggested for these astrocytic progenitor cel

ISSN:0360-4012    

DOI:10.1002/jnr.490370308

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

年代:1994

数据来源: WILEY

摘要:

AbstractInduction of neurite outgrowth from superior cervical ganglia (SCG) by rat lymphoid tissues was studied using a tissue culture model. Neonatal rat SCG were cultured with 6–12‐week‐old rat thymus, spleen, or mesenteric lymph node (MLN) explants in a Martrigel layer, in defined culture medium without exogenous nerve growth factor (NGF). SCG were also co‐cultured with neonatal rat heart (as positive control) or spinal cord (SC; as negative control). To determine whether inflammation affects the ability of lymphoid tissues to induce neurite outgrowth, we also examined MLN at various times after infecting rats withNippostrongylus brasiliensis(Nb‐MLN). In one series of experiments, a single lymphoid tissue explant was surrounded by four SCG at a distance of 1 mm. The extent of neurite outgrowth was determinded by counting the number of neurites 0.5 mm away from each ganglion at several time points. Adult thymus and, to a lesser extent, spleen had strong stimulatory effects on neurite outgrowth from SCG after 12 hr or more in culture. For thymus tissue, this was similar to the positive control heart explants. MLN from normal rats had minimal effect on neurite outgrowth; however, Nb‐MLN showed a time‐dependent enhancement of the neurite outgrowth, maximal at 3 weeks after infection. The relative efficacy of neurite outgrowth induction (heart ≥ thymus ≥ Nb‐MLN ≥ spleen ≥ MLN ≥ SC) was confirmed in a second series of experiments where one SCG was surrounded by three different tissue explants. We then examined the role of 2.5S NGF, a well‐known trophic factor for sympathetic nerves, in the lymphoid tissue‐induced neurite outgrowth. Anti‐NGF treatment of co‐cultures of SCG and heart almost completely blocked the neurite outgrowth. Anti‐NGF also significantly inhibited thymus‐ and spleen‐induced neurite outgrowth, but not as effectively as heart‐induced neuritogenesis (93,80, and 77% inhibition at 24 hr; 86,70, and 68% inhibition at 48 hr for heart, thymus, and spleen, respectively). On the other hand, anti‐NGF inhibited only 8% of neurite outgrowth induced by 3‐week post‐infection Nb‐MLN at 24 hr, and 41% at 48 hr. These data show that several adult rat lymphoid tissues exert neurotrophic/tropic effects. The predominant growth factor in thymus and spleen is NGF, while Nb‐MLN produces factor(s) which is (are) immunologically distinguishable from NGF. These neurotrophic/tropic factors are produced during the reactive lymphoid hyperplasia that forms part of the inflammatory response against the nematode,N. brasiliensis. This suggests the possibility that cytokines produced by lymphocytes or other inflammatory cells may stimulate sympatheti

ISSN:0360-4012    

DOI:10.1002/jnr.490370309

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

年代:1994

数据来源: WILEY

摘要:

AbstractAlthough gangliosides have been reported to enhance recovery from 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced lesions of the substantia nigra, evidence as to whether the administered gangliosides actually reach this or any other site of lesion in the central nervous system (CNS) at which they putatively enhance recovery is lacking. Therefore, studies were carried out to determine the amount of3H‐labeled ganglioside that was accumulated by the brains of MPTP‐treated mice as well as by brains of control mice. No significant difference in the accumulation of3/‐bovine brain gangliosides or3H‐GD1a was seen between lesioned and control brains up to 240 min after injection of the labeled lipids. However, significantly more label was associated with the brains of MPTP‐treated mice compared to controls 120 and 240 min after the injection of3H‐GM1. Analysis of the lipids extracted from the brain of a3H‐GM1‐treated mouse revealed that the majority of label was still associated with3‐GM1, 240 min after its administration. Autoradiography of tissue sections from the brains of MPTP‐treated mice injected with3H‐GM1 showed that label was present in the ventricular spaces of the brain. This observation suggests that the administered gangliosides are present in the cerebrospinal fluid, which indicates that they have the potential to reach the lesioned CNS site at which they putatively enhance

ISSN:0360-4012    

DOI:10.1002/jnr.490370310

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

年代:1994

数据来源: WILEY

摘要:

AbstractUsing quantitative autoradiography, we have characterized the binding properties of the non‐N‐methyl‐D‐aspartate (NMDA) glutamate receptor antagonist [3H]6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) in adult human cerebellum. Saturation experiments revealed [3H]CNQX binding to a single class of sites with similar affinity in the molecular and granule cell layer (Kd= 89.0 ± 6.4 and 83.3 ± 9.9nM, respectively). The maximum number of [3H]CNQX binding sites was much higher in the molecular compared to the granule cell layer (Bmax= 16.2 ± 1.1 and 2.8 ± 0.5 pmol/mg protein, respectively). Inhibition experiments were performed in order to examine the pharmacological profile of [3H]CNQX binding in the molecular layer. [3H]CNQX labeled sites with high affinity for both non‐NMDA agonists, (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) and kainate. Dose‐response curves for inhibition of [3H]CNQX by AMPA and Kainate were biphasic. The potency of AMPA for displacement of [3H]CNQX binding (Ki© 1994 Wiley‐Liss, Inc.:2.8 ± 0.8 nM and 12.5 ± 0.8 μM) was 4‐ to 6‐fold greater than the corresponding potency of kainate (Ki:18.1 ± 5.7 nm and 48.7 ± 9.3 μM). In conclusion, the pharmacological analysis of [3H]CNQX binding in the human cerebellar molecular layer reflects the existence of multiple binding sites of the non‐NMDA receptor that have different aff

ISSN:0360-4012    

DOI:10.1002/jnr.490370311

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

年代:1994

数据来源: WILEY