摘要:

Abstract:One purpose of clinical neurochemistry has been to indicate “activities” of catecholamine systems, by assaying levels of the effector compounds or their metabolites in body fluids such as plasma, cerebrospinal fluid, urine, or microdialysate. This review discusses a new purpose: relating specific catecholaminergic phenotypes to neurogenetic disorders. Distinctive catecholamine patterns in several neurogenetic conditions reflect enzyme deficiencies as direct or indirect effects of gene mutations. These neurochemical patterns can provide potentially important clues to the diagnosis, treatment, and pathophysiology of neurogenetic disorders. Linking genetic abnormalities with molecular mechanisms and clinical manifestations of disease represents a useful new direction in clinical neurochemis

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051781.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:To examine the role of the C terminal tail in H2receptor regulation, three cDNAs, encoding truncated histamine H2receptor mutants (H2T295, H2T307, and H2T341), were constructed and stably transfected in Chinese hamster ovary (CHO) cells. The amino acids before position 307 appear to be necessary for proper receptor transport or folding, as no detectable H2receptor binding of the H2T295 was observed after transfection. Truncation of the C terminal tail by 51 amino acids (H2T307) did not affect the binding properties of H2antagonists and histamine or histamine‐induced signaling. Yet, removal of 17 amino acids generated a mutant receptor (H2T341), which was able to form a ternary complex but was unable to fully activate the Gsprotein on histamine exposure. Agonist‐induced but not the cyclic AMP‐dependent H2receptor down‐regulation was more profound for the H2T307 receptor, indicating that different structural elements of the H2receptor protein are involved in the cyclic AMP‐dependent and independent pathways of H2receptor down‐regulation. Taken together, in this study we identified regions in the C terminal tail of the H2receptor that act as positive and/or negative signals in H2receptor signaling and down

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051791.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Nerve growth factor (NGF) induces persistent p42 and p44 mitogen‐activated protein kinase (MAPK) activity in sympathetic neurones in parallel to its survival‐promoting activity. To investigate whether these MAPK activities are necessary for NGF‐induced survival, we have inhibited NGF‐stimulated p42/p44 MAPK activity over extended periods using the compound 2‐(2′‐amino‐3′‐methoxyphenyl)‐oxanaphthalen‐4‐one (PD98059). Despite attaining up to 95% inhibition of p42/p44 MAPK activity in cultures treated with NGF and PD98059, neuronal survival is maintained undiminished, although a decrease in the density of the neuritic network is observed. Because p21Ras activity is essential for NGF‐induced survival, we conclude that p21Ras‐linked activities other than p42 and p44 MAPKs are responsible for mediating NGF‐dependent survi

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051801.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Previously, we reported that the squid giant axon contains a heterogeneous population of mRNAs that includes β‐actin, β‐tubulin, kinesin, neurofilament proteins, and enolase. To define the absolute levels and relative distribution of these mRNAs, we have used competitive reverse transcription‐PCR to quantify the levels of five mRNAs present in the giant axon and giant fiber lobe (GFL), the location of the parental cell soma. In the GFL, the number of transcripts for these mRNAs varied over a fourfold range, with β‐tubulin being the most abundant mRNA species (1.25 × 109molecules per GFL). Based on transcript number, the rank order of mRNA levels in the GFL was β‐tubulin>β‐actin>kinesin>enolase>microtubule‐associated protein (MAP) H1. In contrast, kinesin mRNA was most abundant in the axon (4.1 × 107molecules per axon) with individual mRNA levels varying 15‐fold. The rank order of mRNA levels in the axon was kinesin>β‐tubulin>MAP H1>β‐actin>enolase. The relative abundance of the mRNA species in the axon did not correlate with the size of the transcript, nor was it directly related to their corresponding levels in the GFL. Taken together, these findings confirm that significant amounts of mRNA are present in the giant axon and suggest that specific mRNAs are differentially transp

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051806.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:To investigate substance P (SP) receptors on an established human astrocytoma cell line (U‐87 MG), [3H][Sar9,Met(O2)11]‐SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti‐SP binding protein antibody. In U‐87 MG and U‐373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U‐87 MG cell membrane‐enriched preparations, the binding of [3H][Sar9,Met(O2)11]‐SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparentKDof 1.15 ± 0.15 nMand aBmaxof 108 ± 9.8 fmol/mg of protein. [3H][Sar9,Met(O2)11]‐SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl‐5′‐imidodiphosphate, but not GDP and GMP, reduced theBmaxwithout changing the affinity of [3H][Sar9,Met(O2)11]‐SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), andLens culinarisagglutinin (LCA)] to determine the nature of carbohydrate chains on the U‐87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U‐87 MG have either a biantennary complex‐type or a high mannose‐type of carbohydrate chain and may be

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051813.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We reported previously that stereoisomers of 1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP), thed‐threoandl‐threoforms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated withl‐ ord‐threo‐PDMP. These isomers exhibited opposite effects on neurite outgrowth:d‐PDMP was inhibitory at concentrations ranging from 5 to 20 µM, whereasl‐PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10–15 µM. Rat neocortical explants were doubly labeled with [14C]serine and [3H]galactose at 15 µMl‐ ord‐PDMP.l‐PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereasd‐PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect ofl‐PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosineN‐acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase (240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action ofl‐PDMP may be ascribable to its stimulatory effect on the biosynt

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051821.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We have previously reported that the polysialoganglioside GT1b suppresses the induction of major histocompatibility complex class I molecules by interferon‐γ in astrocytes. Suppression by GT1b depended on the presence of sialic acid moieties because asialo‐GM1 was not suppressive. In the present report, GT1b was found to act transcriptionally to suppress class I genes because both the interferon‐γ induction of RNA and the activity of class I promoter constructs were inhibited. Furthermore, GT1b suppressed promoter activity through interferon regulatory factor elements, indicating an effect on the transcription activation factor, interferon regulatory factor 1. Interferon‐γ induced interferon regulatory factor 1 within 8 h, and GT1b suppressed this induction. The suppression of interferon regulatory factor 1 by GT1b correlated with the suppression of γ‐activated factor binding at the promoter of the interferon regulatory factor 1 gene. The suppression of γ‐activated factor by GT1b appeared to involve increased protein tyrosine phosphatase activity because treatment of the cells with pervanadate reversed the effect of GT1b on the γ‐activated factor and, correspondingly, phosphotyrosine content. In sum, GT1b displays specific effects on interferon‐γ signaling and negative feedback regulatory m

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051831.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We have previously demonstrated that activation of the Na+‐Ca2+exchanger in the reverse mode causes Ca2+influx in astrocytes. In addition, we showed that the exchange activity was stimulated by nitric oxide (NO)/cyclic GMP and inhibited by ascorbic acid. The present study demonstrates that the Na+‐Ca2+exchanger is involved in agonist‐induced Ca2+signaling in cultured rat astrocytes. The astrocytic intracellular Ca2+concentration ([Ca2+]i) was increased byl‐glutamate, noradrenaline (NA), and ATP, and the increases were all attenuated by the NO generator sodium nitroprusside (SNP). SNP also reduced the ionomycin‐induced increase in [Ca2+]i. The Na‐induced Ca2+signal was also attenuated byS‐nitroso‐l‐cysteine and 8‐bromo cyclic GMP, whereas it was enhanced by 3,4‐dichlorobenzamil, an inhibitor of the Na+‐Ca2+exchanger. Treatment of astrocytes with antisense, but not sense, deoxynucleotides to the sequence encoding the Na+‐Ca2+exchanger enhanced the ionomycin‐induced increase in [Ca2+]iand blocked the effects of SNP and 8‐bromo cyclic GMP in reducing the NA‐induced Ca2+signal. Furthermore, the ionomycin‐induced Ca2+signal was enhanced by removal of extracellular Na+and pretreatment with ascorbic acid. These findings indicate that the Na+‐Ca2+exchanger is a target for NO modulation of elevated [Ca2+]iand that the exchanger plays a role in Ca2+efflux when [Ca2+]iis rais

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051840.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Incubation of cultured hippocampal slices with an inhibitor [N‐CBZ‐l‐phenylalanyl‐l‐alanine‐diazomethyl ketone (ZPAD)] of cathepsins B and L resulted in the degradation of high molecular weight isoforms of τ protein and the production of a 29‐kDa τ fragment (τ29). A τ antibody that is sensitive to the phosphorylated state of its epitopes did not recognize τ proteins or the τ29fragment in slices that had been treated with a protein phosphatase inhibitor. This strongly suggests that the τ fragment was located in an extralysosomal compartment accessible to kinases and phosphatases. τ29exhibited a significant capacity for binding to microtubules and thus has the potential for interfering with normal τ‐tubulin interactions. Three lines of evidence indicated that ZPAD‐induced τ proteolysis was mediated by cathepsin D: (a) slices treated with the inhibitor had markedly elevated levels of cathepsin D in both lysosomal and extralysosomal compartments; (b) co‐incubation of cathepsin D and τ at neutral pH resulted in a loss of intact τ proteins and production of a 29‐kDa fragment; and (c) the lysosomotropic drug chloroquine blocked ZPAD‐induced increases in mature cathepsin D, and this was accompanied by a suppression of ZPAD‐induced τ proteolysis. Changes in lysosomal hydrolases and cytoskeletal perturbations occur during brain aging. The present results suggest that the enzymatic and structural effects are related and, more specifically, are linked by alterations in the concentration and localization of cathepsin D. The τ fragments with microtubule binding capacity generated by cathepsin D could also be a source for the small polypeptides found in association

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051846.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We describe here two types of apoptotic cell death observed in the rat CNS‐derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 mMDBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatase

ISSN:0022-3042    

DOI:10.1046/j.1471-4159.1996.67051856.x

出版商:Blackwell Science Ltd    

年代:1996

数据来源: WILEY