摘要:

AbstractA biologically active fusion protein comprising a short hydrophilic leader peptide fused to the N‐terminus of rat CNTF was generated using commercially available materials. The coding region for rat CNTF was sub‐cloned into the pFLAG‐1 vector and transfected into the JM 109 strain ofE. coli.The transfected cells expressed high levels of the fusion protein (FLAG‐CNTF) following induction by isopropyl β‐D‐thiogalactoside (IPTG). FLAG‐CNTF is expressed as insoluble material that was resolubilized by extraction with guanidine hydrochloride and purified by immuno‐affinity chromatography. Analysis of the purified material by reverse phase HPLC and Western blot analysis indicated that FLAG‐CNTF was composed of two closely related species that are greater than 99% pure after affinity chromatography. The purified FLAG‐CNTF migrated as a 27 KD doublet on SDS polyacrylamide gel electrophoresis. Both bands of the doublet were shown to contain the FLAG peptide and CNTF by Western blot analysis, and amino acid sequence analysis demonstrated a single amino acid sequence corresponding to FLAG peptide and the N‐terminus of CNTF. The purified fusion protein was tested for biological activity using the IMR‐32 human neuroblastoma cell line. Treatment of IMR‐32 cells with FLAG‐CNTF increased the level of choline acetyltransferase (ChAT) in these cells 2–3‐fold over that of control cells in a dose dependent manner. A direct comparison of the effects of FLAG‐CNTF and recombinant human CNTF on IMR‐32 ChAT activity showed that both factors exhibited similar potencies. Our studies demonstrate the use of simple, commercially available, recombinant technology to efficiently generate large quantities of purified, biologically

ISSN:0360-4012    

DOI:10.1002/jnr.490380202

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

年代:1994

数据来源: WILEY

摘要:

AbstractA subset of human anti‐GM1 ganglioside antibodies cross‐reacts with Gal(β1–3)GalNAc bearing glycoproteins in peripheral nerve and spinal cord. The same oligosaccharide determinant is recognized by the lectin peanut agglutinin (PNA) which binds at the nodes of Ranvier in intact peripheral nerve. The Gal(β1–3)GalNAc bearing glycoproteins were isolated using PNA lectin affinity chromatography followed by separation on Western blot, and the proteins were subjected to partial amino acid sequence analysis. Two major PNA binding glycoproteins were identified in peripheral nerve and spinal cord; one had an approximate molecular weight of 120 kD and had sequence homology to the oligodendrocyte‐myelin glycoprotein (OMgp). The other migrated between 70 and 80 kD and had sequence homology to the hyaluronate binding domain of versican, which has been reported to share sequence homology with the 70 kD proteins hyaluronectin and the glial hyaluronic acid binding protein (GHAP). By immunocytochemistry, OMgp was localized to the paranodal region of myelin, and the protein homologous to the hyaluronate binding domain of versican was localized to the nodal gap in peripheral nerve. These PNA binding glycoproteins might be target antigens for autoantibodies in peripheral nerve. © 1994 Wil

ISSN:0360-4012    

DOI:10.1002/jnr.490380203

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

年代:1994

数据来源: WILEY

摘要:

AbstractExperimental diabetic neuropathy includes the non‐enzymatic glycosylation (or glycation) of the major proteins of peripheral nerve myelin. We have used X‐ray diffraction to determine whether such glycation affects myelin membrane structure and interactions in peripheral nerves from experimental diabetic rats. Streptozocin at 60 mg/kg was injected intraperitoneally to induce diabetes; controls were pair‐fed and age‐matched. Animals were sacrificed periodically from 2 weeks to after 1 year. The dissected sciatic nerves were tied off and incubated overnight at room temperature in hypotonic saline of defined pH and ionic strength or in distilled water. Such treatments have been shown to result in systematic changes of myelin period, which can be detected using X‐ray diffraction, and which may indicate alterations in inter‐membrane interactions owing to changes in composition. We observed no differences in repeat periods between control and diabetic nerves at pH 4.0 and 7.4, and ionic strength 0.01, 0.02, 0.06, 0.15, and 0.18; however, we did detect a significant difference (P<.02) in their maximum extent of swelling in distilled water: control nerves showed a period of 292 Å (s.d. 23 Å; n = 12) compared to 272 Å (s.d. 19 Å; n = 11) for diabetic nerves. To determine whether this difference in swelling was due to an alteration in the properties of the apposed, extracellular surfaces of the myelin membranes or to the connective tissue in peripheral nerve, we compared the X‐ray patterns from peripheral nerve myelin isolated by sucrose density gradient centrifugation from sciatic nerves of diabetic and control rats. No difference in the patterns was observed. Since the maximum extent of myelin membrane swelling in peripheral nerves is limited by the collagen, our results suggest that the collagen in the nerves of diabetic rats is less elastic or more densely packed than in the controls, most likely due to N‐glycation‐mediated cross‐linking.

ISSN:0360-4012    

DOI:10.1002/jnr.490380204

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

年代:1994

数据来源: WILEY

摘要:

AbstractDrebrins are developmentally regulated actin‐binding proteins. In this study, we analyzed subcellular distribution of drebrin E in neuroblastoma cells (SHSY5Y) in culture, especially in terms of its relationship to actin filaments. In undifferentiated cells, drebrin E was scattered as flocculus small dots along the stress fibers and also accumulated at adhesion plaques. In parallel with the neuronal differentiation following retinoic acid treatment, drebrin E was accumulated, accompanying filamentous (F) actin, in the submembranous cortical cytoplasm. Similar submembranous localization of drebrins was observed in primary cultured neurons. In the presence of drebrin E F‐actin was more stable against cytochalasin D than F‐actin lacking drebrin E. These results suggest that drebrin E plays a role in neuronal morphological differentiation by changing its subcellular localization with stabilized F‐actin. © 1994 Wiley

ISSN:0360-4012    

DOI:10.1002/jnr.490380205

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

年代:1994

数据来源: WILEY

摘要:

AbstractExposure of individual purified neurofilament (NF) proteins to AlCl3alters their electrophoretic properties in a time‐ and concentration‐dependent manner, as visualized by their failure to migrate into SDS gels. Co‐incubation of purified high (NF‐H) and middle (NF‐M) but not low (NF‐L) molecular weight NF subunits prevents this AlCl3‐induced alteration in electrophoretic migration. This latter finding suggested that specific interactions between NF‐H and NF‐M other than filament formation influenced their interaction with AlCl3. Co‐incubation of the 160 kDa α‐chymotryptic cleavage product of NF‐H (corresponding to the highly phosphorylated C‐terminal sidearm domain) with native NF‐M prevented alteration in subunit electrophoretic migration by AlCl3. By contrast, intact, dephosphorylated NF‐H subunits were unable to prevent AlCl3‐induced alteration of native NF‐M electrophoretic migration. Taken together, these findings suggest that phosphate‐dependent interactions between the sidearm extensions of NF‐H and NF‐M diminish the ability of AlCl3to associate with either subunit in a manner that alters their electrophoretic migration. This interaction of NF‐H and NF‐M sidearms is SDS‐sensitive, while AlCl3‐induced alteration in electrophoretic migration of individual subunits is SDS‐resistant. Addition of SDS to mixtures of NF‐H and NF‐M subunits disrupted the protective effect, and promoted AlCl3‐induced alterations in subunit electrophoretic migration. These findings support and extend the current hypothesis that the ability of aluminum to interact with NF subunits is a function of subunit phosphorylation, assembly, and extent of neurofila

ISSN:0360-4012    

DOI:10.1002/jnr.490380206

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

年代:1994

数据来源: WILEY

摘要:

AbstractSR13/PMP‐22 is a protein that was identified after screening a sciatic nerve cDNA library. Our study focused on comparing the level and pattern of expression of SR13/PMP‐22 protein and RNA. Northern blot analysis revealed that although SR13/PMP‐22 mRNA was present in all nervous tissues and cells studied, levels were at least seven fold higher in the sciatic nerve and the spinal cord. During sciatic nerve postnatal development and maturation, the SR13/PMP‐22 mRNA was detected at 2 days after birth, reached a maximal level at day 24, and decreased to 1/3 of the maximum in adult animals. Nerve transection reduced the level of SR13/PMP‐22 mRNA to less than 5% in the segment distal to the nerve injury. Experiments using in situ hybridization localized the SR13/PMP‐22 mRNA in Schwann cells. Schwann cells present in the vicinity or distal to the nerve cut repressed the signal for the message. In situ hybridization experiments also demonstrated that dorsal root ganglia satellite cells contained the message for SR13/PMP‐22. The SR13/PMP‐22 antisera used in our study showed a complex pattern of staining. As expected, the SR13/PMP‐22 antibody peptide 1 immunoreacted with the sciatic nerve sheath. However, immunocytochemistry of the dorsal root ganglia revealed that the staining was contained in the neuron's cell body and processes and also in satellite cells. We also identified immunoreactive cell bodies and fibers in the spinal cord dorsal horn. Tissue culture studies demonstrated that SR13/PMP‐22 mRNA is induced in NGF treated PC12 but not in C6 glioma cell lines grown under experimental conditions that stimulated cell growth arrest. Our experiments suggest that SR13/PMP‐22 may have some other function(s) in addition to its hypothesized role in peripheral myelination.

ISSN:0360-4012    

DOI:10.1002/jnr.490380207

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe developmental pattern of expression of the G protein αOsubunit and GAP43 were compared by immunohistochemical staining of mouse embryos. Staining for αOand GAP43 was identical and detected throughout the developing nervous system, and the antigens first appeared in neurons at the beginning of neuronal differentiation. GAP43 and αOwere not detected in regions containing only neuroblasts. These observations suggest that αOand GAP43 may not be required for the decision to pass from neuroblast to differentiated neuron, but may play a role in signal transduction during early neuronal development. © 1994 Wiley‐Lis

ISSN:0360-4012    

DOI:10.1002/jnr.490380208

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

年代:1994

数据来源: WILEY

摘要:

AbstractGlutamatergic neurons in the rat stomach were localized immunohistochemically using antibodies against L‐glutamate (L‐Glu) as well as glutamate synthesizing enzyme, glutaminase (GLNase). Myenteric ganglia and nerve bundles in the circular muscle and the longitudinal muscle were found to contain GLU‐ and GLNase‐positive nerve fibers, while submucosa and mucosa were devoid of glutamatergic innervation. The distribution of glutamatergic neurons and their processes in both myenteric ganglia and circular muscle is heterogeneous within the stomach. The effect of L‐Glu on gastric acid secretion was investigated on an everted preparation of isolated rat stomach. L‐Glu at 10−7and 10−8M alone had no effect on acid secretion. It was found that the oxotremorine‐, histamine‐, or gastrin‐stimulated acid secretion was markedly reduced by L‐Glu at 10−8M, whereas L‐Glu had little effect on the acid secretion stimulated by dimethyl‐phenylpiperazinium (DMPP) at this concentration. However, at higher concentration, e.g., 10−7M, L‐Glu also markedly reduced DMPP‐induced acid secretion. Among L‐Glu receptor agonists tested, quisqualic acid (QA) is most potent, followed by kainic acid (KA) and N‐methyl‐D‐aspartic acid (NMDA) in inhibiting oxotremorine‐stimulated acid secretion. Furthermore, this inhibitory effect of L‐Glu on oxotremorine‐stimulated acid secretion is blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), a specific non‐NMDA receptor antagonist.All these results suggest that glutamatergic neurons are involved in the modulation of gastric acid secretion via ionotropic QA/KA receptors, prob

ISSN:0360-4012    

DOI:10.1002/jnr.490380209

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe present investigations were undertaken to examine the susceptibility of type‐2 astrocytes to elevated temperature. Type‐2 astrocytes are much more easily injured by temperature elevation than type‐1 astrocytes. This may be related to cellular redox potential. Type‐1 astrocytes have a greater cytosolic NAD redox potential (i.e., higher NADH:NAD levels) than type‐2 astrocytes as evidenced by a 9‐fold higher ratio of lactate to pyruvate released into the medium by type‐1 astrocytes than type‐2 astrocytes. Heat stress causes the induction of hsp‐72 in both type‐2 and type‐1 astrocytes; however, hsp‐72 protein expression is retained for a longer period of time by the type‐2 astrocyte. A possible basis for the greater sensitivity of type‐2 astrocytes to stress may be a poorer ability to scavenge free radicals. This differential sensitivity of one neural cell type relative to another to elevated temperature may be of significance in understanding the effects of hyperthermia on the developing br

ISSN:0360-4012    

DOI:10.1002/jnr.490380210

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

年代:1994

数据来源: WILEY

摘要:

AbstractThe putative role of growth factors in remyelination was investigated in pure oligodendrocyte (OL) secondary cultures derived from newborn rat brain. These cells form myelin‐like membranes and were used as a model system for toxic attack. A 24 hr treatment with 2.10–5M lysophosphatidylcholine (LPC) induced a loss of 59% of the cells in these cultures, with a 64% reduction in [125I]‐iododeoxyuridine incroporation compared to untreated controls. An absence of processes and myelin‐like sheaths was observed in the remaining cells. Numerous intractoplasmic inclusions were observed on transmission electron microscopy. Immunocytochemical studies with A2B5 monoclonal antibody (mAb), which recognizes oligodendrocyte‐type 2 astrocyte (O‐2A) precursors, OL‐1 mAb (directed against cell surface sulfatides), and anti‐myelin basic protein (anti‐MBP) antibody showed that the entire OL lineage was affected at all stages of maturation. A 3 day treatment with 10 ng/ml basic fibroblast growth factor (bFGF) induced reconstruction of myelin‐like membranes, albeit less compacted than in untreated controls. The doubling in number of cells and the 46% increase in [125I]‐iododeoxyuridine incorporation was due essentially to proliferation of O‐2A progenitors. These results indicate that if bFGF release occurs during demyelination, it may participate in myelin repair mechanisms in the central nervous system.

ISSN:0360-4012    

DOI:10.1002/jnr.490380211

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

年代:1994

数据来源: WILEY