摘要:

AbstractEukaryotic proteins with a carboxyl‐terminal CaaX motif are modified by isoprenylation and subsequently processed by proteolysis of the three terminal amino acids and carboxylmethylation of the exposed cysteine residue. The myelination‐associated 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase (CNP) has a C‐terminal CTII sequence and is isoprenylated; however, no examples of subsequent processing exist when threonine, a polar residue, is located adjacent to the cysteine. Here we show that CNP is capable of being carboxylmethylated in both insect cells and glioma cells. This processing is dependent upon isoprenylation of the cysteine and can be inhibited with the isoprenylated cysteine derivative, N‐acetyl‐S‐farnesyl‐L‐cysteine. Although the role of the methyl group at the C‐terminus of other isoprenylated proteins is not fully understood, modulation of signal transduction pathways is strongly indicated. This modification of CNP may similarly regulate cell biological processes in myelinogene

ISSN:0360-4012    

DOI:10.1002/jnr.490390502

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe presence of the microtubule‐associated protein tau in skin fibroblasts derived from Alzheimer patients and normal controls was investigated using a panel of well‐characterized anti‐tau antibodies against epitopes spanning the tau protein from the amino to the carboxyl end. The antibodies immunolabeled a fine, fibrillar cytoplasmic network in all skin fibroblasts. Disruption of the microtubule network with colchicine did not affect the immunolabeling of the fibrillar network nor did treatment with cytochalasin B known to disrupt the microfilament network. Immunoelectron microscopy with the antitau antibodies revealed colocalization of the label with the 10 nm intermediate filaments. Furthermore, immunoblots found no reactivity against purified vimentin, suggesting that the antibodies recognize an intermediate filament‐associated protein. The findings indicate the presence of tau or a protein with considerable homology to tau in fibroblasts associated with intermediate filaments and not microtubules. © 1994 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490390503

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

年代:1994

数据来源: WILEY

摘要:

AbstractMitogenic effects of fetal calf serum (FCS), platelet‐derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor‐β (TGF‐β), and forskolin to adult mouse Schwann cells were examined by bromodeoxyuridine (BrdU) incorporation and double immunofluorescence for S100 and BrdU. PDGF‐BB, basic FGF, and TGF‐β1 and β2 were all mitogenic for Schwann cells in media containing FCS. Forskolin suppressed the mitogenic activity of these factors. In serum‐free media, PDGF‐BB and bFGF were also mitogenic, but TGF‐β1 and β2 were not. Heparin‐binding fractions of FCS obtained by heparin‐Sepharose chromatography synergized with TGF‐β1 and β2 to produce a mitogenic response. Since PDGF‐BB, acidic FGF, and basic FGF were not detected in these fractions by immunoabsorption and immunoblot assays, the presence of unidentified heparin‐binding molecules in FCS bioactive for adult mouse Schwann cells is

ISSN:0360-4012    

DOI:10.1002/jnr.490390504

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe cellular distribution of synapsin I in chick spinal cord has been examined during embryo development and in cultured neurons from different developmental stages. Using immunocytochemical methods we have observed that synapsin I appears lightly detectable in spinal cord of embryonic day (E)5–E8 embryos when the motor neurons have already established functional contacts with muscle fibers, and increases at E9. Until E8 synapsin I immunoreactivity appeared mainly localized in the gray matter of spinal cord; immunostaining of white matter becomes clearly evident only at E9. These observations indicate that synapsin I expression and possibly its transport to the nerve terminals may be stimulated by sequential signals. The cellular distribution of synapsin I observed in vivo is maintained in E8 and E9 spinal cord neuron cell cultures. In fact, in E8 cultured neurons, synapsin I immunostaining is observed only in the cell body, while in E9 cultured neurons both cell body and fibers are stained. The addition of muscle extracts to E8 cultures induces synapsin I decoration of fibers similar to that observed in E9 cultured neurons. Indeed Western and Northern blot analysis and in situ hybridization demonstrate an increase of synapsin I and its mRNA in spinal cord neurons kept in the presence of muscle extracts. These data suggest that synapsin I expression, as previously reported for other neuronal markers, can be modulated by soluble factors present in target cells. © 1994 Wiley‐Liss,

ISSN:0360-4012    

DOI:10.1002/jnr.490390505

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe cutaneous electroreceptor “tuberous organs” of the lateral line system ofEigenmannia virescenswere studied at light and electron microscopic levels with immunohistochemical and autoradiographic techniques after sectioning of the posterior branch of the lateral line nerve. After deafferentation total degeneration of the sensory cells was observed. The accessory cells of the basal platform, however, remain intact and undergo a process of differentiation. The cytoplasms and nuclei of these cells increase in volume, and the nuclei incorporate tritiated thymidine. In control tuberous organs with intact innervation, tritiated thymidine is absorbed by the nuclei of the elongated epidermal cells surrounding the sensory cavity. The newly formed sensory cells, but not those of the intact organs, are substance P immunoreactive. They have synaptic bars surrounded by vesicles and their free membrane surface is covered with microvilli. The new sensory cells are fully differentiated 35 days after the lateral line was cut. These results demonstrate that in the tuberous organs ofE. virescensnew sensory cells are formed in the absence of an innervation. © 1994 Wiley‐Lis

ISSN:0360-4012    

DOI:10.1002/jnr.490390506

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe effects of ATP (5–500 μM) were evaluated on the proliferation rate of cultured astrocytes by measuring3H‐thymidine incorporation and by flow cytometric analysis of the cell cycle. Determinations after 16 hours showed that ATP present in the culture medium for the whole period caused a dose‐dependent reduction of cell proliferation, while if the exposure to ATP was limited to the first 8 hours, the proliferation was increased (always in a dose‐dependent manner). A time course study of3H‐thymidine incorporation showed that, in the presence of ATP,3H‐thymidine was incorporated at a slower rate than in controls; the replacement of the culture medium with an ATP‐free fresh medium, at the 8th hour, was followed by a3H‐thymidine incorporation occurring at such a fast rate to overshoot the control values. High performance liquid chromatography (HPLC) analysis, carried out to identify purine compounds present in the culture medium during cell exposure to ATP, indicated that more than 95% of the added ATP was metabolized within 1 hr. Conversely, an increase of purine metabolites was measured, this accumulation being greater at the highest concentrations of added ATP. The presence of high levels of extracellular ATP catabolites suggested that these compounds may act on the regulation of cell replication via the different purine receptors. This hypothesis was tested and confirmed by using agonists and antagonists selective for the P1and the P2sites. One hundred μM 2methylthio‐ATP (2MeSATP), a P2Yagonist metabolized as fast as ATP, reproduced effects very similar to the ATP‐induced ones. On the other hand, the nonhydrolisable ATP analogue, adenosine 5′‐(beta, gamma‐imido)‐triphosphate (AMP‐PNP) at 100 μM, induced a mitogenic effect as well as the A2site stimulation. On the contrary, the activation of A1receptors by 5 μM R‐phenyl‐isopropyladenosine (R‐PIA) inhibited astrocyte proliferation; moreover, 100 nM 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), an A1site antagonist, reversed the ATP‐induced inhibition of cell proliferation. These results indicate that exogenous ATP, as a consequence of its rapid extracellular breakdown, exerts a dual influence on astrocyte proliferation by the involvement of both P1and P2Yreceptors. These findings might be relevant to such pathological conditions of the central nervous system (CNS), as seizures, hypoxia or ischemia, in which great amounts of purines released in the brain can influence a reactive astrocyte prolifera

ISSN:0360-4012    

DOI:10.1002/jnr.490390507

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

年代:1994

数据来源: WILEY

摘要:

AbstractOver the past two decades acetylcholinesterase (AChE) has been shown to be present in numerous non‐cholinergic and non‐cholinoceptive tissues. Interestingly, transient expression of AChE in developing nervous tissue corresponds temporally with neuronal migration and neuritic outgrowth. This observation has led our laboratory to investigate a possible novel, non‐cholinergic role for AChE in the development of the nervous system. In a previous study, we demonstrated that the activity of AChE in cultured dorsal root ganglion neurons (DRGN) can be modulated by the substratum. In our current study, we have examined the effects of AChE inhibitor treatment on neuritic outgrowth on the highly permissive substratum MatrigelTMand the less permissive substratum Collagen Type I. DRGN received serial dilutions of the AChE‐specific inhibitor 1,5‐bis‐(4‐allyldimethylammoniumphenyl) pentan‐3‐one dibromide (BW284c51) ranging from 10−4to 10−7M. Results showed that neuritic outgrowth was significantly reduced in DRGN grown on MatrigelTMat 10−5and 10−4M BW284c51, while outgrowth on Collagen Type I was significantly reduced at 10−6, 10−5, and 10−4M concentrations of BW284c51. Inhibitor treatment did not affect cell survival and neuritic outgrowth from BW284c51‐treated cells recovered to control levels after removal of the inhibitor from the medium. In addition, massive spiraling accumulations of 10 nm filaments were observed in the cell bodies of treated neurons, which resemble neurofibrillary inclusions observed in neuropathological diseaes such as Pick's disease. This study demonstrates that AChE inhibitor treatment retards neuritic outgrowth and neuronal migration of cultured DRGN which is accompanied by cytoskeletal abnormalities in the cell body. These data further suggest a novel, non‐cholinergic role for AChE in neural development and r

ISSN:0360-4012    

DOI:10.1002/jnr.490390508

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

年代:1994

数据来源: WILEY

摘要:

AbstractCytochrome P450IIB1,2 (nomenclature according to Nelson et al., DNA Cell Biol 12:1–51, 1993 and Volk et al., Neuroscience 42:215–235, 1991) immunoreactivity (P450‐IR) is associated with astrocytes both in vivo and in vitro. Although they are unevenly distributed throughout the brain with a preference for phylogenetically elder parts, no significant differences between astrocytes prepared from different brain regions were observed in astrocyte cultures. The percentage of strongly immunoreactive astrocytes decreased from 40% after 7 days in culture to 15% after 21 days. Essentially all astrocytes have a low but significant P450‐IR within this interval. Preembedding immunoelectron microscopy revealed peroxidase reaction products on the endoplasmic reticulum and on the outer membranes of mitochondrial and nuclear envelopes. Phenytoin (1 μM) added to the medium for 7 days significantly (1.22‐fold) increased the amount of total P450 in astrocyte homogenates as measured by spectrophotometry. Considerably more immunoreactive cells (1.5‐fold) were found in treated cultures than in controls. © 1994 Wi

ISSN:0360-4012    

DOI:10.1002/jnr.490390509

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

年代:1994

数据来源: WILEY

摘要:

AbstractAlz‐50, a monoclonal antibody originally prepared using Alzheimer brain homogenates, reacts with PHF‐tau and normal tau on immunoblots, and stains specific neuronal populations in sections from Alzheimer's disease brain. Although the Alz‐50 epitope has been mapped to amino acids 2–10 present in all human tau isoforms, minimal Alz‐50 immunoreactivity is present in tissue from control brain, suggesting Alz‐50 binding may be dependent on tau conformational differences. The absence of conclusive results concerning Alz‐50 binding presents the possibility of Alz‐50 immunoreactivity with proteins other than tau. The present study demonstrates Alz‐50 crossreactivity with denatured bovine serum albumin (BSA) and human serum albumin (HSA). Using LA‐N‐5 neuroblastoma cells, BSA from serum‐containing media was present in cell homogenates and was found to be Alz‐50‐reactive on immunoblots. In fact, Alz‐50 (0.1 μg/ml) recognized as little as 78 ng of BSA and 312 ng of HSA. Since Alz‐50 does not recognize native BSA, blocking of immunoblots with 3% BSA did not alter Alz‐50 reactivity with tau from LA‐N‐5 cells. On SDS‐polyacrylamide gels, HSA (∼ 69 kDa) migrates very closely to the pattern of A68 (PHF‐tau) from Alzheimer brain homogenates. Hence, the presence of BSA or other albumins in cell or brain homogenates may be an important concern when using

ISSN:0360-4012    

DOI:10.1002/jnr.490390510

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe concentration of dopamine, and its metabolites 3,4‐dihydroxyphenylacetic and homovanillic acids, as well as serotonin and its metabolite 5‐hydroxyindoleacetic acid, were determined in the retina of two teleosts,C. auratus(goldfish) andE. plumieri(mojarra), and two mammals,R. norvegicus(rat) andO. cuniculus(rabbit). The turnover rate of these monoamines were investigated in the four species by the calculation of the ratio monoamine/metabolite as an indirect index, and in goldfish and rat by the inhibition of the synthesis with α‐methyl‐p‐tyrosine orp‐chlorophenylalanine, by the increase in dopamine or serotonin by the corresponding precursors, 3,4‐dihydroxyphenylalanine or 5‐hydroxytryptophan, and by inhibition of monoaminooxidase with pargyline. The modulation by light and dark stimulation was studied in the goldfish and the rat. Differences in the concentration and turnover rate were observed among the species. Serotonin concentration was higher in the teleosts. The administration of inhibitors of dopamine and serotonin synthesis differentially decreased the levels of the monoamines in the retina of goldfish and rat. The rate of formation of dopamine and serotonin by the corresponding precursors was much higher in the goldfish than in the rat. Pargyline administration decreased 3,4‐dihydroxyphenylacetic and 5‐hydroxyindoleacetic acids at different rates and time dependency in the retina of goldfish and rat. Dopamine and serotonin concentration did not exhibit high modifications by the inhibitor, suggesting the function of regulatory mechanisms or additional effect of pargyline at other sites different from monoaminooxidase. Light stimulation decreased the ratios of dopamine and its metabolites in the retina of goldfish, but not in the rat, probably by a greater release of dopamine with lower uptake capacity in the latter. Goldfish or rat retinal serotonin did not change by light exposure. Homovanillic acid was not detected in either type of retina after the dark period. In the goldfish, there was an increase in dopamine/3,4‐dihydroxyphenylacetic acid ratio by the darkness; in the rat, this ratio decreased, which might be related to the release of dopamine. Retinal serotonin turnover rate was accelerated in the goldfish by dark stimulation. The present comparative results support a differential regulation of monoamine levels among species, and will be useful for further characterization of the regulatory mechanisms related to light and dark exposure.

ISSN:0360-4012    

DOI:10.1002/jnr.490390511

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

年代:1994

数据来源: WILEY