Neuroprotocols最新文献

{"title":"α-Thrombin-Stimulated 1,2-Diacylglycerol Formation: The Relationship between Phospholipid Hydrolysis and Protein Kinase C Activation","authors":"K. Leach, D. Raben","doi":"10.1006/NCMN.1993.1042","DOIUrl":"https://doi.org/10.1006/NCMN.1993.1042","url":null,"abstract":"Phosphoinositide hydrolysis plays an important role in cellular signaling because it results in increased levels of calcium and diacylglycerols (DGs), which in turn activate protein kinase C (PKC). Agonist-induced hydrolysis of phosphatidylcholine (PtdCho) has been demonstrated, which also results in DG formation. However, it has not been clearly established whether PtdCho-derived DGs activate PKC in intact cells. We addressed this question directly, using α-thrombin stimulation of IIC9 fibroblasts as a model system. We show that DG produced from phosphoinositide, but not PtdCho hydrolysis, is associated with the activation of PKC. In addition, the methods used to quantify and chemically analyze agonist-induced changes in lipid levels, as well as PKC activation, are reviewed in detail.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"79 6 1","pages":"91-102"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77300131","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Use of U-73122 as an Inhibitor of Phospholipase C-Dependent Processes","authors":"J. E. Bleasdale, S. Fisher","doi":"10.1006/NCMN.1993.1046","DOIUrl":"https://doi.org/10.1006/NCMN.1993.1046","url":null,"abstract":"1-[6-[[17β-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]1H-pyrrole-2,5-dione (U-73122) is an aminosteroid that was identified initially as a potent inhibitor of platelet activation by receptor-specific agonists. U-73122 inhibits receptor-coupled generation of inositol 1,4,5-trisphosphate (but not cyclic AMP) and intracellular mobilization of Ca 2+ in a variety of cell types. U-73122 inhibits phosphoinositide-specific phospholipase C (PI-PLC) activity in cell-free systems, but exhibits little or no direct inhibition of phospholipases A2 and D. Structure-activity analysis revealed that the maleimide group of U-73122 is essential, but not sufficient, for inhibitory activity. The succinimide analog of U-73122 (U-73343) has negligible inhibitory activity and is a useful control compound. On the basis of information derived from the use of U-73122 in a variety of cell types, procedures for storing, dissolving, and presenting U-73122 to cells are recommended. While knowledge of the mechanism of action of U-73122 would extend the utility of this compound, U-73122 has already been employed successfully to examine PI-PLC involvement in a variety of cellular processes. The application of U-73122 in an investigation of muscarinic receptor sequestration in SK-N-SH neuroblastoma cells is illustrated.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"11 1","pages":"125-133"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85793948","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Analysis of Changes in the Molecular Species of Glycerolipids in Cultured Cells during Signal Transduction","authors":"Lee Chunghee,&nbsp;Hajra Amiya K.","doi":"10.1006/ncmn.1993.1041","DOIUrl":"https://doi.org/10.1006/ncmn.1993.1041","url":null,"abstract":"<div><p>A method for the quantitative analysis of the molecular species of glycerolipids present In biological samples has been described. 1,2-Diacyl-<em>sn</em>-glycerol, either isolated from biological samples or enzymatically generated from phosphoglycerides, is benzoylated at the <em>sn</em>-3 position and then subjected to reverse-phase (C₁₈-silica) HPLC to separate the molecular species of different hydrophobicitles. An internal standard (1,2-distearoyl-<em>sn</em>-glycerol) is used to identify and quantify the various species eluted from the reverse-phase column. Examples are given for the quantitative analysis of molecular species and precursor-product relationships of glycerolipids generated In SK-N-SH neuroblastoma cells after stimulation of the cell-surface muscarinic acetylcholine receptors.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"3 2","pages":"Pages 83-90"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1993.1041","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72117725","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessment of Neuronal Phosphoinositide Turnover and Its Disruption by Lithium","authors":"Challiss R.A.J.,&nbsp;Jenkinson S.,&nbsp;Mistry R.,&nbsp;Batty I.H.,&nbsp;Nahorski S.R.","doi":"10.1006/ncmn.1993.1047","DOIUrl":"https://doi.org/10.1006/ncmn.1993.1047","url":null,"abstract":"<div><p>The ability of lithium to interfere with signal transduction pathways that involve neurotransmitter receptor activation of phosphoinositide turnover has been proposed as a potential mechanistic explanation of the therapeutic actions of lithium in manic-depressive illness. Noncompetitive inhibition of inositol monophosphatase by submillimolar concentrations of lithium deprives active neurons of endogenously generated <em>myo</em>-inositol. If this deficit cannot be compensated for by uptake of extracellular <em>myo</em>-inositol, then the ability of the cell to synthesize and maintain inositol phospholipid pools will be compromised. Here we describe methods for the investigation of the phosphoinositide cycle, with particular emphasis on methods that have been used to highlight the complex actions of lithium to disrupt activation of this important signal transduction pathway by neurotransmitters.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"3 2","pages":"Pages 135-144"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1993.1047","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72082857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signaling by Neurotrophic Factors: Activation of Phosphoinositide 3-Kinase by Nerve Growth Factor","authors":"A. Carter, C. Downes","doi":"10.1006/NCMN.1993.1044","DOIUrl":"https://doi.org/10.1006/NCMN.1993.1044","url":null,"abstract":"Abstract Phosphoinositide 3-kinase (PI 3-kinase) is thought to play an important role in mitogenic signal transduction initiated by both receptor tyrosine kinases and nonreceptor tyrosine kinases. We have shown recently that this enzyme is also potently activated by nerve growth factor (NGF) in PC12 cells, a model cell line in which application of NGF induces differentiation to a neuronal phenotype. This finding implicates PI 3-kinase as a component of the signal transduction pathways required for neuronal development and survival under the control of NGF. Whether PI 3-kinase is activated by other neurotrophic factors is not yet clear, but it is possible that this enzyme plays a pivotal role in the development and maintenance of the mammalian nervous system. In this paper we detail methods that can be used to monitor the regulation of PI 3-kinase and the amounts of its lipid products in stimulated cells. The general utility, advantages, and pitfalls of these experimental approaches are discussed.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"22 1","pages":"107-118"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80170068","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Analysis of Changes in the Molecular Species of Glycerolipids in Cultured Cells during Signal Transduction","authors":"Chunghee Lee, A. Hajra","doi":"10.1006/NCMN.1993.1041","DOIUrl":"https://doi.org/10.1006/NCMN.1993.1041","url":null,"abstract":"Abstract A method for the quantitative analysis of the molecular species of glycerolipids present In biological samples has been described. 1,2-Diacyl- sn -glycerol, either isolated from biological samples or enzymatically generated from phosphoglycerides, is benzoylated at the sn -3 position and then subjected to reverse-phase (C 18 -silica) HPLC to separate the molecular species of different hydrophobicitles. An internal standard (1,2-distearoyl- sn -glycerol) is used to identify and quantify the various species eluted from the reverse-phase column. Examples are given for the quantitative analysis of molecular species and precursor-product relationships of glycerolipids generated In SK-N-SH neuroblastoma cells after stimulation of the cell-surface muscarinic acetylcholine receptors.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"3 1","pages":"83-90"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83922456","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signaling by Neurotrophic Factors: Activation of Phosphoinositide 3-Kinase by Nerve Growth Factor","authors":"Carter A.Nigel,&nbsp;Downes C.Peter","doi":"10.1006/ncmn.1993.1044","DOIUrl":"https://doi.org/10.1006/ncmn.1993.1044","url":null,"abstract":"Abstract Phosphoinositide 3-kinase (PI 3-kinase) is thought to play an important role in mitogenic signal transduction initiated by both receptor tyrosine kinases and nonreceptor tyrosine kinases. We have shown recently that this enzyme is also potently activated by nerve growth factor (NGF) in PC12 cells, a model cell line in which application of NGF induces differentiation to a neuronal phenotype. This finding implicates PI 3-kinase as a component of the signal transduction pathways required for neuronal development and survival under the control of NGF. Whether PI 3-kinase is activated by other neurotrophic factors is not yet clear, but it is possible that this enzyme plays a pivotal role in the development and maintenance of the mammalian nervous system. In this paper we detail methods that can be used to monitor the regulation of PI 3-kinase and the amounts of its lipid products in stimulated cells. The general utility, advantages, and pitfalls of these experimental approaches are discussed.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"3 2","pages":"Pages 107-118"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1993.1044","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72117728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Regulation of Glycosyl-phosphoinositides in Mammalian Cells","authors":"Pearl Cathryn,&nbsp;Saltiel Alan R.","doi":"10.1006/ncmn.1993.1045","DOIUrl":"https://doi.org/10.1006/ncmn.1993.1045","url":null,"abstract":"<div><p>Glycosyl-phosphoinositide molecules have both structural and functional roles in mammalian cells. These glycophospholipids can serve as membrane anchors for cell surface proteins or as precursors for the generation of second messengers in hormone action. Methodology for analysis of the synthesis and metabolism of these molecules is outlined. Tissue culture cells are used for experiments involving labeling with radioactive precursors. After exposure to hormones, glycosyl-phosphoinositides and their metabolites can be analyzed by a combination of thin-layer and high-performance liquid chromatography.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"3 2","pages":"Pages 119-124"},"PeriodicalIF":0.0,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1993.1045","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72117729","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Laser-Driven Photochemical Induction of Spinal Cord Injury in the Rat: Methodology, Histopathology, and Applications","authors":"Watson Brant D.,&nbsp;Holets Vicky R.,&nbsp;Prado Ricardo,&nbsp;Bunge Mary Bartlett","doi":"10.1006/ncmn.1993.1032","DOIUrl":"https://doi.org/10.1006/ncmn.1993.1032","url":null,"abstract":"<div><p>Experimental modeling of spinal cord injury is based mostly on mechanical effects, such as the impact of a weight dropped on the exposed spinal cord. The development of the resultant lesion is influenced by many interactive factors, e.g., the efficiencies of momentum and energy transfer to the cord and their profiles in time, and consequently the histopathologic reproducibility of the lesion is often inconsistent. We describe here a recoilless method that avoids these complications (as well as laminectomy) inherently. The vascular endothelium is injured photochemically, yielding chiefly small-vessel thrombosis and associated vasogenic edema sufficient to generate spinal cord necrosis to predetermined, reproducible degrees. This model is thus intended to simulate the secondary response of the vasculature to mechanical injury In the absence of hemorrhage. The most efficient version of this technique utilizes argon-dye laser excitation of the photosensitizing dye rose bengal at its 562-nm absorption maximum in tissue. With the laser beam focused in the shape of a thin (0.3-mm) line transverse to the spinal column at T8, a narrow zone of necrosis is initially produced. Within 1 week this initial zone expands in volume (length, 6-7 mm) to create a space that, when cleared of cellular debris, is suitable for cell Implantation. In cross section, striking features are the sharp horizontal demarcation between necrosed and viable tissue and the uniform progression of lesion depth as a function of Irradiation time. The necrotic region is bordered dorsally and laterally by a thin rim of viable tissue except at the beam focus; starting at 5 days there is evidence of demyelination in this peripheral region. By 14 days, myelination by oligodendrocytes and Schwann cells begins near this rim; large numbers of Schwann cells enter the dorsal cord at the epicenter, and myelinated axons occupy previously degenerated areas. By 2 months, the initial necrotic area begins to diminish, flattening laterally into clefts. Large, empty cavities develop secondarily with luminal surfaces that are smoothly contoured, as seen by electron microscopy, in contrast to the relatively irregular border of the initial necrotic lesion. Many of these morphological attributes mirror those observed after contusion injury. We anticipate that the uniformity and reproducibility of the photochemical lesion will prove useful in conducting statistically efficient tests of strategies, such as the administration of drugs, trophic factors, or transplanted cells, which are proposed to improve outcome after spinal cord injury.</p></div>","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"3 1","pages":"Pages 3-15"},"PeriodicalIF":0.0,"publicationDate":"1993-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1006/ncmn.1993.1032","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72122200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transgenic Mouse Approaches for Analysis of the Nervous System","authors":"H. Friedman, J. Julien","doi":"10.1006/NCMN.1993.1039","DOIUrl":"https://doi.org/10.1006/NCMN.1993.1039","url":null,"abstract":"Abstract The transgenic mouse technology offers the opportunity to introduce or to replace genetic information in the mouse germ line. We discuss the advantages and limitations of the various methods of creating transgenic animals as well as their potential applications to the study of gene regulation and function in the nervous system. We present applications of reporter genes, such as LacZ, whose activity is detectable in situ with histochemical staining, to elucidate the molecular signals controlling the spatial and temporal expression of genes during neurogenesis. The transgenic system offers a unique way of examining in vivo the mechanisms modulating gene expression during neural regeneration. Once transgenic mouse lines are established with a variety of DNA constructs, the cis-regulatory elements involved in up- or down-regulation of a transgene can be examined in vivo after axotomy. Different strategies for realizing the gain or loss of gene activity can provide information on the function of gene products. To direct expression of genes to different cell types in the nervous system, different promoters are now available. Examples of transgenic mice with overt phenotypes are presented, namely, mice exhibiting motor neuronopathy as a result of expression of a neurofilament transgene and mice with aberrant sprouting of sensory axons in the spinal cord as a result of constitutive expression of a NGF construct. The approaches used to abolish partial or complete gene function are addressed. Recent reports of gene knockout experiments with phenotypes such as abnormal sensory innervation (p75NGFR), learning deficits (α-calcium-calmodulin kinase II), or loss of specific CNS regions during development (Wnt-1) illustrate the great potential of the gene-targeting approach for analyzing complex neural functions and for deriving new animal models for human neurological disorders.","PeriodicalId":100951,"journal":{"name":"Neuroprotocols","volume":"135 1","pages":"69-80"},"PeriodicalIF":0.0,"publicationDate":"1993-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75824395","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}