摘要:

AbstractThe proteolipid proteins (PLP and DM20) are major constituents of CNS myelin, but how they are delivered to and organized within the oligodendrocyte plasma membrane is incompletely understood. We have expressed both PLP and DM20 singly or together in a host cell line, HeLa. In either DM20 or PLP transfectants, at early time points (24 hours), the expressed proteins are found within intracellular compartments. In DM20 transfectants, the protein is delivered to the plasma membrane by 48 hours. In HeLa cells, PLP remains intracellular when expressed in the absence of DM20; only when it is coexpressed with DM20 is it transported to the plasma membrane. In cotransfectants, PLP can also be localized to organelles involved in both the protein biosynthetic and the endocytic pathways. Since, in HeLa cells at least, the delivery of PLP to the plasma membrane is facilitated by the coexpression of DM20, we suggest that the two proteins interact intracellularly to form a complex. In some PLP/DM20 cotransfectants, the proteolipids are concentrated in regions of cell‐cell contact. The regional accumulation of these proteins at cell‐cell interfaces is highly reminiscent of the behavior in transfected cells of another myelin protein, Po, and certain cadherin polypeptides, both of which have readily demonstrable membrane adhesive properties. Our data suggests that at certain stoichiometric ratios, proteolipids can become stablized at cell surfaces to form adhesive bonds. © 1994 Wiley‐Lis

ISSN:0360-4012    

DOI:10.1002/jnr.490370502

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

年代:1994

数据来源: WILEY

摘要:

AbstractDelineating the properties and functions of the major central nervous system myelin proteins has been the focus of intensive research for decades. For PLP, this task has been confounded by its unusual properties, the complexity of the cellular membrane in which it resides, and the absence of a functional assay for the protein. The development of new experimental paradigms in which to study PLP may shed fresh light on the properties and functions of this intrinsic membrane protein. In the present communication we have used indirect, double label, immunofluorescence, and confocal microscopy to examine the distribution of PLP in Cos‐7 cells transfected with an expression vector bearing the human PLP cDNA. Our results show that PLP is synthesized in the rough endoplasmic reticulum of transfected cells and passes through the Golgi apparatus to the cell surface. These results are consistent with previous studies showing PLP reaches the cell surface by transport through the secretory pathway. Levels of PLP at the cell surface are modest, most likely because protein deposited in this compartment can be endocytosed and subsequently transported to perinuclear lysosomes. Similar results are reported in the companion communication by Sinoway et al. (J Neurosci Res, 37:551–562, 1994). Using transfected HeLa cells they show that DM20 alone and PLP coexpressed with DM20 assume appropriate conformations for transport to the cell surface.The presence of PLP in subcellular compartments beyond the endoplasmic reticulum in Cos‐7 cells indicates that the protein achieves a conformation appropriate for transport in the absence of other oligodendrocyte‐specific factors; however, accumulation of large amounts of PLP in the cytoplasmic membrane compartment may require interactions with such glial‐specific factors. Thus, the transfection paradigm described herein should prove a useful tool for investigating the folding and sorting of wild type and mutant forms of PLP as well as its membrane topology and posttranslational p

ISSN:0360-4012    

DOI:10.1002/jnr.490370503

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe primary structure of the proteolipid protein (PLP) from the central nervous system (CNS) myelin of mammals is highly conserved with only three amino acid differences between the mouse, rat, dog, bovine, and human proteins. Furthermore, within a particular species no polymorphisms in the protein have been identified. Recent interest has focused on the targeting of PLP in oligodendrocytes and the role that mutant forms of this protein play in generating dysmyelinating or hypomyelinating diseases. We previously expressed the human cDNA encoding PLP in transiently transfected Cos‐7 cells and characterized the subcellular distribution of the protein in this simple heterologous system. In the current study we have used the same paradigm to examine the effect of five missense mutations in the PLP gene on processing of the encoded protein. The mutations chosen span the carboxy‐terminal half of PLP and encompass that part of the protein in which most mutations have been identified. Our results show that transport of all mutations examined was arrested in the secretory pathway at an early stage, causing the mutant proteins to accumulate in the endoplasmic reticulum. Thus, a common mechanism of protein misfolding and failure of PLP to reach the cell surface of oligodendrocytes rather than the inability of the mutant protein to perform some crucial function at the cell surface may be responsible for the diseases caused by many PLP mutations. Our results, together with those of others, prompt us to speculate that the pathobiology observed in PLP mutants may result from oligodendrocyte cell death caused by the accumulation of misfolded protein in the endoplasmic reticulum. This speculation is consistent with the observations that oligodendrocytes bearing misfolded PLP, as in thejimpymutant, proliferate but die rapidly while oligodendrocytes from PLP deletion survive and produce a myelin‐like membrane which lacks PLP. © 1994 Wiley‐L

ISSN:0360-4012    

DOI:10.1002/jnr.490370504

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

年代:1994

数据来源: WILEY

摘要:

AbstractCell‐surface heparan sulfate proteoglycans (HSPGs) are potential mediators of neuronal cell adhesion, spreading, and neurite outgrowth on various extracellular matrix molecules. One possible site of HSPG attachment is a heparin binding domain of fibronectin, which is present in the synthetic peptide FN‐C/H II. In this study, HSPGs extracted from embryonic rat spinal cord by detergent were purified by ionexchange chromatography, gel filtration, and affinity chromatography on an agarose column coupled with FN‐C/H II conjugated to ovalbumin (OA). Heparitinase treatment of the iodinated HSPG fraction led to the appearance of a major protein core with a molecular size of 72 kDa, as determined by reducing SDS‐PAGE. The intact proteoglycan has a molecular size of approximately 150–165 kDa, containing heparan sulfate glycosaminoglycan chains of about 10–15 kDa. Anti‐HSPG antibodies recognized the 72 kDa core protein by immunoblotting, and stained the surface of spinal cord neurons, oligodendrocytes, and a subset of astrocytes. These results identify a cell‐surface HSPG that may mediate neuron‐substratum or neuron‐glia interactions in embryonic central nervous system. ©

ISSN:0360-4012    

DOI:10.1002/jnr.490370505

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe distribution of dorsal root ganglion (DRG) cell sizes that show changes in preprotachykinin (PPT) gene expression and substance P (SP) levels following axotomy was examined using RNA blot analysis, in situ hybridization histochemistry, and immunocytochemistry. PPT mRNA was induced in medium‐sized (1,000–2,000 μm2) and large‐sized (>2,000 μm2) cells in the DRG after axotomy. There was a 165% increase in the number of labeled cells after sciatic transection and a 260% increase after spinal nerve transection which results in axotomy of all the cells in the ganglion. The further increase after spinal nerve transection suggests that the induction occurred in axotomized neurons. PPT mRNA label was also present in a reduced number of small (<1,000 μm2) cells after axotomy. SP immunoreactivity was also induced in medium‐and large‐sized cells and reduced in small‐sized cells. Our findings suggest that the expression of the PPT gene and SP is differentially regulated in different subpopulations of DRG neurons after axotomy and is consistent with the hypothesis that tachykinins may be important in both sensory transmission and regeneration. © 1994

ISSN:0360-4012    

DOI:10.1002/jnr.490370506

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

年代:1994

数据来源: WILEY

摘要:

AbstractSince synthesis of myelin components has been seen to be stimulated by cAMP in both oligodendrocytes and Schwann cells we have begun investigating the specific sequence(s) in the 5′ flanking region of the myelin basic protein (MBP) gene that are responsible for the induction of MBP transcription by cAMP. Using stably transfected cell lines containing various deletions of the MBP promoter directing the bacterial chloramphenicol acetyltransferase (CAT) gene we have identified a region of the MBP gene that is inhibitory to stimulation by increased cAMP levels. This inhibition can be overcome by pretreating the cells with 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) for 48 hr. The effects on MBP gene expression modulated by TPA and cAMP involve altered DNA‐protein interactions in the 5′ end of the MBP promoter. The effect of TPA also appears to be mediated by down‐regulation of protein kinase C. © 1994 W

ISSN:0360-4012    

DOI:10.1002/jnr.490370507

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

年代:1994

数据来源: WILEY

摘要:

AbstractMouse sciatic nerves were transected and 3 hr to 16 days later proximal segments were removed and homogenized. Supernatants of these segments or of normal sciatic nerves were added to Schwann cells maintained in Dulbecco's modified Eagle's medium (DMEM) + 15% fetal calf serum (FCS). After 6 days, Schwann cells were solubilized and the protein content was measured using a Bio‐Rad (Melville, NY) protein assay. Samples containing the same amounts of protein were then applied to microtiter plates and the laminin content was determined by enzymelinked immunosorbent assay (ELISA). Lysates of cultures treated with 24 hr proximal segment supernatants contained significantly higher levels of laminin than those prepared from other intervals, from distal segments, or from control nerves. Increased surface and cytoplasmic anti‐laminin immunoreactivity also was found in Schwann cells treated with 24 hr supernatants. To identify the source(s) of this effect, proximal segments removed 24 hr after transection were bisected; supernatants were prepared from each half and tested. Significant increases in laminin production were produced by supernatants from both halves. When supernatants from proximal and distal halves were compared, the latter produced significantly higher laminin levels. Electron microscopic examination of both halves showed that distal halves contained sprouting neurites and growth cones ensheathed by Schwann cells which had a basal lamina and resembled those seen during development and regeneration. Proximal halves appeared normal. Schwann cell proliferation also was compared in supernatant‐treated cultures by using a bromodeoxyuridine (BrdU) ELISA. The 24 hr and 2 day supernatants increased Schwann cell proliferation significantly; 12 hr, 4 day, and 8 day supernatants produced smaller increases. Our observations suggest that axons undergoing early regenerative changes are one of several possible sources of substance(s) in our proximal segment supernatants which increased Schwann cell proliferation and laminin production. © 1994 Wiley‐L

ISSN:0360-4012    

DOI:10.1002/jnr.490370508

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

年代:1994

数据来源: WILEY

摘要:

AbstractWe recently reported evidence implicating a superior colliculus‐derived chondroitin sulfate proteoglycan (SCCP) in the trophic support of cultured retinal ganglion cells (Schulz et al., 1990). In the present work we show preparations of the SCCP to be reactive with an antibody (CS‐56) to chondroitin sulfate types A and C and with the HNK‐1 antibody. Reaction with the HNK‐1 antibody allowed us partially to purify the native proteoglycan by immunoaffinity chromatography. HNK‐1 reactive material was further processed by a combination of molecular sieve chromatography in the presence of 4M guanidine HCL followed by anion exchange chromatography to yield a product that migrated electrophoretically as a single band in polyacrylamide gel with an apparent molecular weight of not less than 400k. The SCCP, when added to a fully defined culture medium, maintained the survival of the vast majority (80%) of the ganglion cells over a 16 hr culture period with 86% of these cells showing a profusion of processes; few ganglion cells (10%) survived in the absence of the proteoglycan. Electrophoretic analysis of nonreduced preparations of the molecule did not reveal any low molecular weight silver stained components that may have remained associated with the molecule after guanidine HCL treatment. However, two bands corresponding to molecular weights of around 60 and 80 k were reproducibly obseved on polyacrylamide gels following electrophoresis of the molecule in the presence of β‐mercaptoethanol. Our findings provide further evidence suggesting a role for a chondroitin sulfate proteoglycan carrying the HNK‐1 epitope in the trophic support of central neurones. © 1994

ISSN:0360-4012    

DOI:10.1002/jnr.490370509

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe evolution of the neuropeptide Y (NPY) family of peptides has been unclear despite sequence information from many vertebrates. We describe here two NPY‐related peptides deduced from cDNA clones of the river lamprey (Lampetra fluviatilis), a cyclostome providing one of the best models of a primitive vertebrate brain. One peptide corresponds to NPY as it has 83% identity to human NPY and its mRNA is expressed in the lateral brainstem, dorsal spinal cord and retina. The second lamprey peptide corresponds anatomically to peptide YY (PYY) as its mRNA is found in gut cells and in medial brainstem neurons. Its sequence is 60–70% identical to both PYY and NPY of mammals. These data suggest that the gene duplication leading to NPY and PYY had already occurred in the ancestral vertebrate 450 million years ago. The expression of the presumed PYY homolog in both gut and central nervous system indicates that PYY has served the dual role as a hormone and a neuropeptide from an early stage in vertebrate evolution. The similarities in the location of NPY‐ and PYY‐expressing cells between lamprey and mammals suggest that the functions of these peptides may have been conserved. © 1994 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490370510

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

年代:1994

数据来源: WILEY

摘要:

AbstractMicrodialysis‐perfusion of the urethane‐anesthetized rat hippocampus was performed to assess the effects of acidosis on extracellular amino acids. Perfusion with Krebs‐Ringer bicarbonate buffer at pH 6.9 produced a selective decrease in taurine. A further reduction of pH to 6.4 induced diminished glutamate levels. The CI−/HCO3−exchange inhibitor 4,4′‐diisothiocyano‐2,2′‐disulfonic acid stilbene (DIDS) did not affect interstitial taurine during perfusion, but there was a rebound increase in taurine levels upon withdrawal of the agent. In contrast, glutamate concentrations were elevated during DIDS administration, and decreased upon reperfusion with standard buffer. The reduction of extracellular taurine and glutamate concentrations caused by low pH was inhibited by DIDS. The results suggest that taurine and glutamate uptake and/or release in vivo is pH‐dependent, and that the effects of acidosis possibly are mediated by the CI−/HCO3−antiporter. The decrease in extracellular glutamate brought about by low pH may have pathophysiologic implications in conditions associated with disturbed pH homeostasis such as cerebral ischemia and spreading depressio

ISSN:0360-4012    

DOI:10.1002/jnr.490370511

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

年代:1994

数据来源: WILEY