摘要:

AbstractTo explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular, In this program, propensities for the secondary structure conformations as well as physical‐chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two‐dimensional map of the correlation coefficients.Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that theconservedresidues included most of the amino acids which were predicted to form the α or β conformations, while thealteredresidues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of β propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed α‐β‐α folding. To trace the immunoglobulin fold along the P0 sequence, we compared the β propensity curve of P0 with that of the immunoglobulin M603, whose three‐dimensional structure has been determined. We propose that the flat β‐sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra β‐fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces and may account for a smaller extracellular space as measured by x‐ray diffraction. A computer search of the myelin protein sequences for functional motifs revealed sites for N‐glycosylation, phosphorylation, nucleotide binding, and certain enzyme activities. We note especially that there are potential nucleotide binding sites in proteolipid protein (PLP), MBP and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP). This is consistent with the experimental observations that PLP acts like an ionophore or proton channel when reconstituted into planar lipid bilayers, MBP binds GTP, and CNP catalyzes in vitro the hydrolysis of 2′,3′ nucle

ISSN:0360-4012    

DOI:10.1002/jnr.490280102

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

年代:1991

数据来源: WILEY

摘要:

AbstractAxons and axon terminals are widely believed to lack the capacity to synthesize proteins, relying instead on the delivery of proteins made in the perikaryon. In agreement with this view, axoplasmic proteins synthesized by the isolated giant axon of the squid are believed to derive entirely from periaxonal glial cells. However, squid axoplusm is known to contain the requisite components of an extra‐mitocliondrial protein synthetic system, including protdn factors, tRNAs, rRNAs, and a heterogeneous family of mRNAs. Hence, the giant axon could, in principle, maintain an endogenous protein synthetic capacity. Here, we report that the squid giant axon also contains active polysomes and niRNA, which hybridizes to a riboprobe encoding murine neurofilament protein. Taken together, these findings provide direct evidence that proteins (including the putative neuron‐specific neurofilament protein) are also synthesized de novo in the axonal compartm

ISSN:0360-4012    

DOI:10.1002/jnr.490280103

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

年代:1991

数据来源: WILEY

摘要:

AbstractAfter retinoic acid treatment, a large percentage of cells of the human embryonal carcinoma cell line NT2/D1 differentiate into neuronal cells. We demonstrate here that the differentiated cells, but not the undifferentiated cells, contain high levels of neurofilament mRNA. We have also measured mRNA, protein, and activity levels of two kinases, cAMP‐dependent protein kinase (PKA) and protein kinase C (PKC), in order to explore the role of protein kinases in the establishment of the differentiated state. RNA levels for the catalytic (Cα and Cβ) subunits of PKA increased after differentiation. Total PKA activity levels increased 7‐fold in the differentiated cells. Parallel with this, a rise in the level of catalytic subunit protein occurred. A 12‐fold induction of Type 2 (β) PKC mRNA levels was observed after neuronal differentiation. Increases in PKC activity and in Type 2 (β) and Type 3 (α) PKC protein levels also accompa nied differentiation. These changes in PKA‐ and PKC‐specific RNA levels and enzyme activity may be necessary for production and maintenance of the differentiated state

ISSN:0360-4012    

DOI:10.1002/jnr.490280104

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

年代:1991

数据来源: WILEY

摘要:

AbstractLithium salts are the most effective agents used in treating manic‐depressive illness. It has been suggested that lithium's therapeutic efficacy could be due to an inhibitory effect on either inositol phospholipid (IP) and/or cyclic nucleotide metabolism. We have investigated the effect of lithium on these two signal transduction pathways in PC12 pheochromocytoma cells by studying a common effector target, expression of the fos protooncogene. We find that lithium, at therapeutic doses, has an augmenting effect on phosphatidylinositol (PI)‐mediated fos expression induced by activating a muscarinic cholinergic pathway, whereas it has no effect, at tenfold the therapeutic dose, on fos expression induced by receptor or postreceptor activators of cyclic adenosine monophosphate (cAMP). The lithium augmenting effect is also observed when the cells are treated with phorbol esters, which directly activate protein kinase C (PKC), suggesting that the level of lithium's interaction with the IP pathway is at the postreceptor level. We also show that phorbol esters induce extensive down regulation of subsequent cholinergic and phorbol ester responsiveness as well as heterologous down regulation of cAMP responses. Treatment of down‐regulated cells with lithium leads to an enhanced responsiveness when cells are rechallenged with agonists that activate PKC but not by agonists that stimulate cAMP. We also show that carbamazepine, another antimanic agent, has an inhibitory effect on cAMP‐mediated fos but no effect on the IP pathway. The opposite effects of lithium and carbamazepine on two critical transducing systems suggest a model for the antimanic action of these

ISSN:0360-4012    

DOI:10.1002/jnr.490280105

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe distribution of proteolipid protein (PLP) and myelin basic protein (MBP) was examined in differentiating Oligodendrocytes of primary and secondary mouse brain cell cultures by single‐ and double‐label indirect immunofluorescence. In primary cultures, MBP and PLP were differentially located in Oligodendrocytes. MBP became concentrated as fine punctate dots lining the edges of processes and as coarse grains in flattened sheet‐like structures. PLP was distributed diffusely throughout cell bodies and processes but was limited to the perimeter of sheets and some processes within sheets. To compare the detailed distribution of PLP and MBP in the absence of underlying cells, a simple method for the growth of isolated Oligodendrocytes in secondary cultures was developed. Cells were maintained in primary culture for 39‐41 days, harvested by scraping, enriched for Oligodendrocytes, and plated at low cell density. After 1 week, isolated Oligodendrocytes had developed long processes and large flattened membranous sheets. MBP and PLP were differentially localized in these cell structures. The sheets contained fine‐grained patches of MBP, which were surrounded by networks of MBP−processes. In contrast, PLP was initially seen throughout the cell bodies and processes. In older cultures, PLP became strikingly concentrated in curvilinear membranous profiles. The observations show that PLP and MBP aie differentially located in cultured mouse Oligodendrocytes. Further‐more, the precise distribution of these myelin‐specific antigens is dependent on cul

ISSN:0360-4012    

DOI:10.1002/jnr.490280106

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

年代:1991

数据来源: WILEY

摘要:

AbstractServeral synaptic parameters, previously shown to undergo alterations with changes in the internal and external environment, were examined in the visual system of light/dark‐ and dark‐reared postnatal and adult rats. Animals were raised in either 14hr light/10 hr dark (Lt/Dk) or in total darkness (Dk). The specific synaptic parameters in the superficial layers of the superior colliculus (SC), visual cortex (laminae II/III and IV) (VC), and, as contro, the auditory cortex (laminae II/III and IV) (AC), examined during the postnatal period (i.e., postnatal days 7,14,21, and 28) and in the adult (i.e., day 56) included:(1) mean number of synapses, (2) mean synaptic length, (3) percentages of perforated postsynaptic densities, (4) percentages of asymmetric and symmetric synapses, (5) percentages of dendritic, spinous, anmd somatic synapses, and (6) percentages of synapses with positive, negative, or no curvature. Developmental patterns in rats rearded in normal lighting conditions were noted. Specifically, in the SC and VC of Lt/Dk animals, the number of synapses increased up to postnatal day in the mean number of synapses increased; no significant changes in the mean number of synapses between PND =28 and 56 were detected in any of the areas examined. Changes in synaptic length in the SC and VC were not observed during postnatal development or in the adult in any of the aforementioned brain areas. Low percentages of postsynaptic densities (PSDs) were found at all time points and in all brain areas during the postnatal period. Increases in perforated PSDs were seen at PND = 56 compared to PND = 28 in the VC. In the VC and AC, there was decrease in symmetric synapses with age. Asymmetric synapses were prevalent in all brain areas at PND = 28 AND 56. dendritic synapses predominated in the SC, while spinous synapses were the preponderant type in the VC and AC during postnatal development and in the adult, A decrease in the percentage of spinous synapses in the SC was observed at PND = 56 vs. PND = 28. A decrease in the percentage of negatively curved synapses with age and a trend toward a concomitant increase in the percentage of positively curved synapses were seen in all brain areas during development and in the adult. Quantitative analyses of the SC and VC tissues examined from all postnatal animals demonstrated no significant differences between Lt/Dk and Dk animals in all the synaptic parameters measured. On the other hand, similar analyses of adult synapses in the SC and VC showed a significantly smaller mean number of synapses in rats dark‐reared to adulthood as compared to those raised in normal lighting conditions. Dark‐rearing had no effect on synaptic number at any time point in the auditory cortex, indicating that this is a specific light effect. Therefore, the effects of visual deprivation on synaptic numbers occur during early adulthood and not during postnatal deve

ISSN:0360-4012    

DOI:10.1002/jnr.490280107

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

年代:1991

数据来源: WILEY

摘要:

AbstractPlasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.:Journal of Neurochemistry55:602–610, 1990). The present study reports the expression and localization of plasmolipin in primary glial cultures and secondary oligooendrocyte cultures. Double‐label immunofluoresccnce showed that plasmolipin was expressed by galactoccrebroside (GC)‐positive oligodendrocytes, but was absent from astrocytes, characterized by their positive staining for glial fibrillary acidic protein (GFAP). At 1 week in culture plasmolipin staining was relatively weak in the cell body of some of the GC‐positive cells. During the following 3 weeks in culture plasmolipin staining of Oligodendrocytes gradually increased and was present in the cell body, its plasma membrane, and all the processes. However, the plasmclipin antibodies did not stain regions of the flat membrane sheets. Western blot analysis of homogenatcs from primary glial cultures showed that plasmolipin levels gradually increased during the first 5 weeks in culture. We conclude that the presence of plasmolipin in myelin is a result of its expression by Oligodend

ISSN:0360-4012    

DOI:10.1002/jnr.490280108

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

年代:1991

数据来源: WILEY

摘要:

AbstractThe beta‐amyloid precursor protein (APP) is a membrane‐bound glycoprotein which has been proposed to play a role both as a growth factor and a mediator of cell adhesion. Using the Ncuro‐2A neuroblastonia cell line, we have investigated the capacity of APP to mediate neural cell adhesion. The cells express the protein at a high level, the immunohistochemical staining pattern at the level of the membrane having a punctate pattern. Fab' fragments of antibodies to the extracellular portion of the molecule were found to inhibit cell binding to a collagen substrate, but not to laminin, fibronectin, or poly‐I‐lysine. Fab' fragments of antibodies to the nerve cell adhesion molecule N‐CAM also inhibited binding of Neuro‐2A cells specifically to collagen. This inhibition of cell‐surface binding was accompanied by a repression of neurite outgrowth in differentiating cells in the presence of antibodies. APP antibodies also inhibited neuron‐neuron and neuron‐glial binding, but not glial‐glial cell adhesion. These data suggest that the APP, which is expressed primarily on differentiated neuronal cells, may play a role in the mediation of both cell‐cell and

ISSN:0360-4012    

DOI:10.1002/jnr.490280109

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

年代:1991

数据来源: WILEY

摘要:

AbstractWe applied serum‐free cell culture methods that allow extended proliferation of mouse astrocyte precursor cells to the multipassagc culture of embryonic human brain cells. Cells were cultured in nutrient medium supplemented with insulin, transferrin, epidermal growth factor, fibreblast growth factor, heparin, high‐density lipoprotein, and fibronectin. Cultures were maintained for a maximum of 70 population doublings before proliferation ceased. The cells synthesized glial fibrillary acidic protein, an astrocyte marker, and expression of this protein was increased by incubation of the cells with transforming growth factor beta or serum. These results identify extracellular factors important for proliferation and differentiation of embryonic human astrocytes and provide a controlled system for multipassage cult

ISSN:0360-4012    

DOI:10.1002/jnr.490280110

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

年代:1991

数据来源: WILEY

摘要:

Abstract1,25‐dihydroxyvitamin D3(1,25‐(OH)2D3), a metabolically active form of vitamin D, is shown to increase in a dose‐dependent manner the cellular pool of NGF mRNA in murine L‐929 fibroblasts cultured in a serum‐free medium. This effect can be detected as early as 3 hours after 1,25‐(OH)2D3addition and persists for at least 28 hours. It is accompanied by an enhancement of the amount of NGF‐protein secreted in the culture medium. Since the proto‐oncogenec‐fosappears involved in the regulation of the NGF gene (Mocchetti et al.:Proceedings of the National Academy of Sciences of the United States of America86:3871‐895, 1989; Hengerer et at:Proceedings of the National Academy of Sciences of the United States of America87:3899‐3903, 1990), the effect of 1,25‐(OH)2D3onc‐fosexpression was analysed and compared to that elicited by other inducers of the NGF gene, serum (Wion et al: FEBS‐ Letters 189:37‐41, 1985) and phorbol 12‐myristate 13‐acetate (PMA) (Wion et al:FEBS Letters262:42‐44, 1990). Addition of serum or PMA to L‐929 cells was; rapidly followed by a transient activation of thec‐fosgene. In contrast,c‐fostranscripts remained undetected in the presence of 1,25‐(OH)3D3The failure to find any evidence of c‐fos expression suggests that 1,25‐(OH)2D3could enhance the pool of NGF mRNA by a mechanism independent of thec‐fospathway. The effective concentration range, as low as 10−10M raises the possibility that 1,25‐(OH)2D3could represent one of the serum factors that might contribute to the regulation of the NGF gene in vitro and possibly in vivo. This suggests a possible therapeutic i

ISSN:0360-4012    

DOI:10.1002/jnr.490280111

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

年代:1991

数据来源: WILEY