摘要:

AbstractAmong the factors which influence neuronal morphology, the degree of substrate adhesivity has been suggested to play an important role in the growth and guidance of neurites. The present study was undertaken to investigate apparently contradictory results relating substrate adhesivity to the extent of neurite outgrowth. By using substrates coated with different concentrations of polyornithine to vary adhesivity, we could show that intermediate levels of neuron‐to‐substrate adhesive strength favored neurite outgrowth more than substrates of high or low adhesivity. However, when neurons were plated on substrates derived from the extracellular matrix, the strength of neuronto‐substrate adhesion was important for the growth of dendrite‐like minor neurites, but not for the extension of axon‐like major neurites, which grew independently of adhesive forces. On substrates of the cell adhesion molecule L1, growth of both major and minor neurites was adhesion‐independent. Finally, in the presence of tenascin added to the culture medium, neurite growth was inhibited irrespective of the adhesivity of the substrate and the presence of substrate‐bound extracellular matrix molecules or L1. These observations suggest that intermediate forces of adhesivity favor neurite growth in general, but that purely adhesive forces can be dominated by specific molecular instructions which differentially affect growth of major and minor neurites in positive and negative ways. © 1995 W

ISSN:0360-4012    

DOI:10.1002/jnr.490420202

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

年代:1995

数据来源: WILEY

摘要:

AbstractIn an attempt to identify genes associated with Wallerian degeneration and peripheral nerve regeneration we have performed differential hybridization screening of a cDNA library from crushed rat sciatic nerve (7 days postlesion) using radioactively labeled cDNA prepared from poly(A)+ RNA of normal vs. crushed nerve. Screening of 5,000 randomly selected colonies yielded 24 distinct clones that were regulated following nerve injury. Fifteen of the differentially expressed sequences could be classified as induced, whereas 9 sequences appeared to be repressed at 1 week postcrush. Sequencing and computer‐assisted sequence comparison revealed 3 classes of regulated cDNA clones representing (1) novel gene sequences (8 clones) including 3 transcripts containing a repetitive “brain identifier” (ID) element; (2) identified genes (7 clones) with previously undetected expression in the peripheral nervous system (PNS), such as apolipoprotein D, peripheral myelin protein 22kD (PMP22), SPARC (secreted protein, acidic and rich in cysteine), sulfated glycoprotein SGP‐1, apoferritin, decorin, and X16/SRp20; and (3) identified genes (9 clones) with known expression in the PNS including, e.g., the myelin protein P0, γ‐actin, vimentin, a‐tubulin, chargerin II, and cytochrome c‐oxidase subunit I. Northern blot and polymerase chain reaction analyses with RNA from crushed and transected nerve demonstrated that sequences with related function, like the group of myelin genes, cytoskeleton genes, genes involved in RNA processing and translation, in lipid transport or energy metabolism showed closely related temporal patterns of expression during nerve degeneration and regeneration. Finally, we compared the differentially expressed genes identified at 7 days after crush injury (this investigation) with the regulated sequences isolated previously by De Leon et al. (J Neurosci Res 29:437‐488, 1991) from a 3 day postcrush sciatic nerve cDNA library. © 199

ISSN:0360-4012    

DOI:10.1002/jnr.490420203

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

年代:1995

数据来源: WILEY

摘要:

AbstractGrowth factors are key elements in the process of neural cell differentiation. We examined the effects of classical mitogens on neural precursor cells, by culturing mouse cells of the embryonic (13.5 days postcoitum) mesencephalon and treating them with epidermal growth factor (EGF), transforming growth factor‐β (TGF‐β), basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and transforming growth factor‐β (TGF‐ β). Our initial results show that EGF, TGF‐α or bFGF, but not NGF or TGF‐β, induced general proliferation of the cultured cells, followed by formation of colonies. Combinations of these three growth factors suggest that most cells with the capacity to form colonies responded to EGF, TGF‐α, or bFGF. The number of colonies increased significantly when EGF, but not TGF‐α, was used in combination with bFGF. Furthermore, a population responding only to EGF + bFGF was detected in the dorsal mesencephalon. The colony‐forming activity of bFGF was dependent on insulin, but bFGF and insulin cooperation was indirect since we could not observe colony formation in subcultures of cells derived from colonies, even in the presence of insulin. Cells obtained from our colonies displayed neuronal and glial morphology and expressed markers of both neurons and astrocytes; nestin, a marker of neural precursor cells was also expressed in the majority of colonies. Growth factors also influenced neuronal maturation; the best neurite outgrowth was obtained from cells derived from bFGF‐induced colonies cultured in the presence of EGF + bFGF. These data indicate the existence of neural precursor cells in the embryonic mesencephalon that respond differentially to growth fact

ISSN:0360-4012    

DOI:10.1002/jnr.490420204

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

年代:1995

数据来源: WILEY

摘要:

AbstractSerum‐free mouse embryo (SFME) cells derived in a defined serum‐free medium have been cultured for more than 200 generations and display properties of neural progenitor cells. SFME cells express the neuroepithelial stem cell marker nestin in defined serumfree medium. Exposure of SFME cells to transforming growth factor beta (TGF‐β) or serum decreases nestin expression and induces the astrocyte marker glial fibrillary acidic protein, suggesting that SFME cells differentiate into astrocytes upon exposure to TGF‐β or serum. We examined the expression by SFME cells of the functional central nervous system (CNS) astrocyte marker glutamine synthetase (GS). GS activity is induced in SFME cells upon exposure to TGF‐β or serum. The induction of GS activity was dose‐ and time‐dependent and was reversible. Retinoic acid, hydrocortisone, and dibutyryl cyclic AMP also induced GS expression. The induction of GS activity was accompanied by an increase in the level of GS mRNA and protein. This work provides further evidence that SFME cells represent neural progenitor cells which differentiate into functional astrocytes upon exposure to TGF‐β or serum. ©

ISSN:0360-4012    

DOI:10.1002/jnr.490420205

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe cell surface sugar determinants (CSSD) were examined in C6 glioma cells in cultures at different conditions of growth by peroxidase conjugates of the lectins: peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), Helix pomatia agglutinin (HPA), wheat germ agglutinin (WGA), lentil agglutinin (LCA), laburnum bork agglutinin (LABA), and lotus agglutinin (TPA). It was found that the cells bound more intensively WGA, LCA, and RCA compared to PNA, HPA; the weakest staining was provided by LABA and TPA. Binding intensity for PNA significantly increased after pretreatment of the cells with neuraminidase. This indicates that a part of the β‐D‐galactose residues on the surface membrane of C6 glioma cells is covered by sialic acid. The process of sialization was increased during the culturing of C6 glioma cells. Addition of cis‐DDP or dBcAMP to cultures growing in medium with 10% of CS increased the number of Gal residues which are not covered by sialic acid.The expression of β‐D‐galactose (Gal), N‐acetyl‐Dgalactosamine (NAcDGal), and fucose (Fuc) residues appeared to be most responsive to changes in growth conditions and degree of cell differentiation. The expressions of N‐acetyl‐D‐glucosamine (NAcDGIc) and mannose (Man) residues were high and seems did not depend on changing of the conditions of culturing.In C6 glioma cells cultures in which the rate of cell division, formation of the cell processes, and adhesiveness of the cells to the substratum were reduced by growing cells in MEM +, expression of β‐Gal, NAcDGa1, and Fuc was considerably reduced. The decrease of expression of R‐Gal, NAcDGaI, and Fuc on the surface of cell membrane was more pronounced in MEM + with 1% of CS than in MEM + with 10% of CS. In DbcAMP and cis‐DDP treated cultures, grown in medium with 1% serum, in which cell division was inhibited without obvious changes in cell adhesiveness to the substratum, binding of PNA and HPA was increased due to higher expression of β‐Gal and NAcDGaI. From these observations it was concluded that the pattern of expression of sugar residues on the cell surface varies according to the biological state of the cells and are easily affected by tissue culture con

ISSN:0360-4012    

DOI:10.1002/jnr.490420206

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

年代:1995

数据来源: WILEY

摘要:

AbstractElk is a member of the eph family of receptor‐like tyrosine kinases. Although its function is unknown, elk is postulated to play a role in nervous system development. Using Northern analysis, we examined the developmental regulation of RNAs encoding elk, and several ligands for the eph family of RTKs, the LERKs. Expression of elk, LERK‐1, and LERK‐2 RNAs is high in all regions examined in the embryonic and postnatal rat brain and decreases to low levels with age. One exception is the adult olfactory bulb which continues to express a moderate level of LERK‐2. In contrast, moderate LERK‐4 expression was limited to the developing hippocampus and cerebral cortex. These data indicate that elk and some of the LERKs may play a role in nervous system development, maintenance, and/or regeneration. © 1995 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490420207

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe effects of a series of protein kinase inhibitors on nerve growth factor (NGF)‐dependent and NGF‐independent neurite outgrowth in PC12 cells have established an ordered relationship among those protein kinases sensitive to down regulation by bryostatin, stimulation by staurosporine, inhibition by sphingosine, or inhibition by 6‐thioguanine (6‐TG). Quantitation of the biphasic staurosporin effects on NGF‐induced neurite outgrowth (Hashimoto and Hagino: J Neurochem 53:1675‐1685, 1989) gave an IC50of 2‐4 nM for inhibition and an EC50of 15‐20 nM for induction of neurite extension. Both sphingosine and 6‐TG inhibited neurite outgrowth induced by staurosporine and basic fibroblast derived growth factor (bFGF), as well as by NGF; therefore, sphingosine‐ and 6‐TG‐sensitive protein kinase steps occur after the convergence of the NGF, bFGF, and staurosporine signal pathways. Down regulation of protein kinase C by bryostatin chronic treatment, which inhibits NGF‐ and bFGF‐induced neuritogenesis (Singh et al.: Biochemistry 33:542‐551, 1994), did not inhibit the staurosporine‐induced neurite outgrowth. Thus, the bryostatin‐sensitive protein kinase C must occur subsequent to the convergence of the bFGF and NGF pathways, but before (or parallel to) staurosporine initiation of neurite outgrowth. In contrast, low concentrations of phorbol myristoyl acetate (PMA) or bryostatin, which activate protein kinase C activity, enhanced the staurosporine‐ or NGF‐induced neurite extension. These data indicate that stimulation of one or more protein kinase C isozymes can synergistically interact with the signaling pathway to increase the rate of neuritogenesis. Inhibition by 5‐7.5 nM staurosporine acted rapidly to arrest and decrease development of neurites up to 24 hr after NGF treatment, as did K252a and NGF ployclonal antibody addition. Our cellular data support the concept that staurosporine acts to inhibit the NGF receptorTrk(Nye et al.: Mol Biol Cell 3:677‐686, 1992), but that downstream steps can be activated by the higher concentration of staurosporine to bypassTrkand lead to neurite generation. Effects of staurosporine, 6‐TG, and sphingosine on c‐fos gene induction with or without NGF were not correlated with the generation of neurites. The sequence of protein kinases sensitive to these effectors appears to be in the order (but not consecutive) bryostatin, staurosporine,

ISSN:0360-4012    

DOI:10.1002/jnr.490420208

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

年代:1995

数据来源: WILEY

摘要:

AbstractThy 1.2 is a well‐known major cell surface glycoprotein of the central nervous system (CNS). However, the regulation of the expression of this molecule as well as its function are yet to be determined. To approach these problems we studied the synthesis of the molecule in the developing cerebellum of wild‐type and staggerer mutant mice. We found the appearance of a [35S]‐methionine‐labeled band detected with specific Sepharose 4B‐bound monoclonal antibodies (Mabs). The Thy 1.2 activity increases progressively from postnatal day 9 (P9), reaching the highest rate at P12, subsequently decreasing sharply at P13, and remaining relatively low up to P16 in the wild type. Comparison of these data to the rates of total protein synthesis reveals a selective developmental regulation of Thy 1.2 expression, at least at the translational level. This correlates quite well with the timing of synaptic stabilization between parallel fibers and Purkinje cell dendritic spines. Furthermore, at P12 Thy 1.2 protein is preferentially located in the synaptosomal fraction. The parallel fiber:Purkinje cell synapsis is not stabilized in the staggerer mutant mouse. At P12 Thy 1.2 synthesis is 30% of the wild type, indicating that the translational regulation of Thy 1.2 is altered in the staggerer mutation. © 1995 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490420209

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

年代:1995

数据来源: WILEY

摘要:

AbstractThe present work describes time‐dependent changes in the content of corticotropin‐releasing hormone (CRH), adrenocorticotropin (ACTH), and β‐endorphin (β ‐EP) in the hypothalamus (HT) and anterior pituitary (AP) and in the concentration of ACTH and β‐EP in the plasma during the 17β estradiol (E2) benzoate (E2B)‐induced luteinizing hormone (LH) surge in ovariectomized cynomolgus monkeys. Monkeys were euthanized at 0, 30, 48, 72, and 96 hr post‐E2B. HT and AP were rapidly dissected, extracted in 2 N acetic acid containing 1 mM phenylmethane sulfonyl fluoride at 4°C, and centrifuged at 18,000g for 30 min. Peptide concentrations were measured in the supernatant by specific radioimmunoassays (RIAs). In the HT, there were significant (P<0.05) decreases in ACTH and β ‐EP content by 30 hr post‐E2B and a significant (P<0.05) decrease in HT CRH content 48 hr post‐E2B. Thereafter, CRH, ACTH, and β‐EP content increased up to 72 hr post‐E2B. In the AP, there was an almost linear decrease in the CRH content through 48 hr post‐E2B followed by a marked 20‐fold (P<0.01) increase in the AP CRH content at 72 hr post‐E2B, which corresponds to the time of the descending arm of the LH surge. The patterns of ACTH and β‐EP content were very similar in the AP, while that of CRH differed markedly. In contrast, in the HT CRH, ACTH, and β‐EP profiles were very similar. Significant (P<0.05) increases in circulating levels of ACTH, β‐EP, and cortisol were evident at 30 hr (all 3 hormones), 48 hr (β‐EP and cortisol), and 72 hr (cortisol) post‐E2B, which corresponds with the time of decreased hypothalamic content of CR1I, ACTH, and β‐EP. These results suggest that there may be a marked activation of the hypothalamo‐anterior pituitary‐adrenal axis during the negative and positive feedback phases of the E2B‐induced LH

ISSN:0360-4012    

DOI:10.1002/jnr.490420210

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

年代:1995

数据来源: WILEY

摘要:

AbstractTo clarify the controversy concerning the role of catecholaminergic systems in regulating hypothalamic corticotropin‐releasing factor (CRF) secretion, we assessed the direct effects of norepinephrine and epinephrine, alone and in association with mixed α and β antagonists on hypothalamic CRF secretion. An in vitro rat mediobasal hypothalamus perifusion system was used, in which CRF secretion from a single explant was evaluated by a specific radioimmunoassay. We found that norepinephrine stimulated CRF secretion, with peak effects at 10‐8M concentration, whereas epinephrine had no effect on CRF secretion. The effect of norepinephrine was antagonised by the mixed a antagonist phentolamine and by the mixed β antagonist propranolol.We conclude that norepinephrine, but not epinephrine, stimulate hypothamic CRF secretion via a and β receptors. The data support the idea that the central noradrenergic systems are excitatory upon CRF‐41 secretion when acting directly at the hypothalamic level. © 1995 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490420211

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

年代:1995

数据来源: WILEY