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SUBCELLULAR-DISTRIBUTION OF 5-HYDROXYTRYPTAMINE IN RAT-BRAIN DURING DEVELOPMENT - EFFECT OF DRUGS AND FASTING

JOURNAL OF NEUROCHEMISTRY 1975年第6期24卷 1143-1150页

作者： TISSARI, AH RAUNU, EM UNIV HELSINKI DEPT PHARMACOL00170 HELSINKIFINLAND

Abstract— Distribution of brain 5-HT content between the high-speed supernatant and particulate fractions under normal and experimental conditions was studied in postnatal and adult rats. In adult and 35-day-old rats the 5-HT content of the supernatant fraction was about 25% of that of the total homogenate and significantly higher than that in 1, 7 and 21-day-old rats. In 1-day-old rats fasting caused an increase of 100% in the turnover, 50% in the content and no alteration in the subcellular distribution of brain 5-HT, which suggests that under normal conditions 5-HT stores may be filled near to capacity. After 5-hydroxytryptophan administration, the 5-HT content of the adult rat brain increased 3-fold and that of the supernatant fraction to 35% of 5-HT content of the total homogenate. In postnatal rats, the brain 5-HT content rose to an adult level and the supernatant 5-HT percentage to a markedly higher than adult level, probably because of the known higher than adult 5-hydroxytryptophan decarboxylase activity of brain capillaries. Administration of tranylcypromine to adult rats caused a 2.6-fold increase of brain 5-HT content and a slight increase of the supernatant 5-HT percentage. At various times after the administration of the MAO inhibitors (tranylcypromine or pargyline) and fasting to the 1-day-old rats, brain 5-HT content increased 4, 5 and 7-fold, respectively, and the supernatant 5-HT rose consistently and, as in the adult, to about 30% of the 5-HT content of the total homogenate. After pargyline following reserpine pretreatment, the 5-HT content of the adult and 1-day-old rat brain increased to 2–3 times the control level and that of the supernatant fraction to about 40% of the 5-HT content of the total homogenate. The adult values for 5-HT in the particulate fraction of the 1-day-old rats after the drug treatments are in sharp contradiction to the low endogenous 5-HT content and known lack of nerve terminals and synaptic vesicles in their brains, and

关键词： 5-羟色氨酸/药理学衰老动物新生脑/药物作用脑/生长和发育脑/代谢禁食帕吉林/药理学利血平/药理学血清素/代谢亚细胞部分/药物作用亚细胞部分/代谢反苯环丙铵/药理学动物大鼠

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Intracellular distribution of a cytoplasmic progesterone receptor mutant and of immunophilins cyclophilin 40 and FKBP59: effects of cyclosporin A, of various metabolic inhibitors and of several culture conditions

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JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 1999年第4-6期70卷 219-228页

作者： Lebeau, MC Jung-Testas, I Baulieu, EE Hop Bicetre INSERM U33 F-94276 Le Kremlin Bicetre France Hop Bicetre INSERM U488 F-94276 Le Kremlin Bicetre France

The effect of cyclosporin A (CsA) on the intracellular distribution of a mutated NLS minus rabbit progesterone receptor (PRm) and the receptor-associated immunophilins, cyclophilin 40 (Cyp40) and FKBP59, was tested in Lcl3 cells by indirect immunofluorescent staining. PRm, which is cytoplasmic in absence of progesterone, is shifted to the nucleus by the hormone as well as by CsA, but not by FK506 or Rapamycin [I. Jung-Testas. M.-C. Lebeau, E.E. Baulieu. C.R. Acad. Sci. Paris 318 (1995) 873-878]. However the time course of nuclear import due to CsA and its sensitivity to N-ethyl maleimide (NEM) and to a calmodulin inhibitor (W7) was different from those observed for the hormonal effect. Cyp40 in Lcl3 cells is localized mainly in the nucleoli. CsA treatment increased nucleolar staining, while NEM and W7 caused it to decrease;after actinomycin D (1 mu M) nucleolar staining of Cyp40 disappeared. FKBP59 is mainly cytoplasmic and concentrated in the perinuclear region, never in the nucleoli. CsA, actino D and W7 treatment did not influence FKBP59 localization. In serum-deprived medium FKBP59 was cytoplasmic, but when the culture medium was enriched (20% serum, insulin and EGF) FKBP59 became perinuclear and hsp 86 was partly shifted to the nucleus, but PRm remained cytoplasmic. CsA has an effect on PRm distribution, while it does not influence Cyp40 and FKBP59 localization. In presence of actino D the labelling of Cyp40 disappears from the nucleoli, while the distribution of PRm and FKBP59 is unaffected. Growth factors influence FKBP59 but not PRm or Cyp40. These results suggest that these proteins shuttle independently and that their association is transient. (C) 1999 Elsevier Science Ltd. All rights reserved.

关键词：钙调蛋白/拮抗剂和抑制剂载体蛋白质类/代谢亲环素类环孢菌素/药理学细胞质/代谢更生霉素/药理学乙基马来酰亚胺/药理学热温度嗜免疫剂/代谢免疫抑制剂/药理学 L细胞(细胞系) 诱变肽基脯氨酰异构酶/代谢酚苄明/药理学孕酮/药理学受体孕酮/遗传学受体孕酮/代谢重组蛋白质类序列缺失西罗莫司/药理学亚细胞部分/药物作用亚细胞部分/代谢磺胺类/药理学他克莫司结合蛋白质类转染动物小鼠兔

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Inhibition of β₂-adrenergic and muscarinic cholinergic receptor endocytosis after depletion of phosphatidylinositol bisphosphate

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 1999年第2期290卷 603-610页

作者： Sorensen, SD Linseman, DA McEwen, EL Heacock, AM Fisher, SK Univ Michigan Mental Hlth Res Inst Neurosci Lab Ann Arbor MI 48109 USA Univ Michigan Dept Pharmacol Ann Arbor MI 48109 USA

Recent evidence supporting a role for phosphoinositides in the endocytosis of phospholipase C-coupled receptors has prompted an investigation of whether there exists a similar requirement for the internalization of adenylyl cyclase-linked receptors. When 1321N1 astrocytoma cells, which possess both muscarinic cholinergic receptors (mAChRs) that couple to phospholipase C and beta-adrenergic receptors (beta(2)-ARs) linked to adenylyl cyclase, were pretreated with wortmannin (WT) at a concentration known to inhibit phosphatidylinositol 4-kinase activity, the labeling of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2) was reduced. Stimulation of phosphoinositide breakdown by activation of mAChRs in WT-pretreated cells led to a further depletion of PIP2. As previously demonstrated for SH-SY5Y neuroblastoma, inclusion of WT inhibited the endocytosis of mAChRs in 1321N1 cells by >85%. In contrast, the internalization of beta(2)-ARs was only partially (similar to 30%) prevented. However, when the concentration of PIP, was further reduced by exposure of WT-pretreated 1321N1 cells to a muscarinic agonist, the endocytosis of beta(2)-ARs was substantially inhibited (>70%). Lower concentrations of WT (100 nM) that were sufficient to fully inhibit phosphatidylinositol 3-kinase activity had no effect on either phosphoinositide synthesis or receptor endocytosis. The results indicate that the agonist-induced endocytosis of an adenylyl cyclase-linked receptor such as the beta(2)-AR, like that of the phospholipase C-coupled mAChR, is dependent on the synthesis of phosphoinositides and, in particular, that of PIP2.

关键词： 1-磷脂酰肌醇3-激酶/拮抗剂和抑制剂肾上腺素能β受体拮抗剂/药理学雄二烯类/药理学钙/代谢钙信号/药物作用细胞质/药物作用细胞质/代谢电泳聚丙烯酰氨凝胶胞吞作用/药物作用酶抑制剂/药理学免疫印迹法磷脂酰肌醇4 5-二磷酸/拮抗剂和抑制剂磷脂酰肌醇磷酸盐类/拮抗剂和抑制剂放射配体测定受体肾上腺素能β2/拮抗剂和抑制剂受体毒蕈碱/药物作用亚细胞部分/药物作用亚细胞部分/代谢肿瘤细胞培养的动物人类大鼠

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Subcellular alterations induced by UV-oxidized low-density lipoproteins in epithelial cells can be counteracted by α-tocopherol

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PHOTOCHEMISTRY AND PHOTOBIOLOGY 2000年第1期71卷 97-102页

作者： Masella, R Straface, E Giovannini, C Di Benedetto, R Scazzocchio, B Viora, M Cantafora, A Malorni, W Ist Super Sanita Dept Metab & Pathol Biochem I-00161 Rome Italy Ist Super Sanita Dept Ultrastruct I-00161 Rome Italy Ist Super Sanita Dept Foods I-00161 Rome Italy Ist Super Sanita Dept Immunol I-00161 Rome Italy

Oxidized LDL (ox-LDL) have been involved in the pathogenesis of several human diseases including dermatological pathologies, Oxidative modification of low-density lipoproteins (LDL) is accompanied by both extensive degradation of its polyunsaturated fatty acids and production of lipoperoxides, These highly reactive products induce an intracellular oxidative stress with a variety of cytotoxic effects. In order to evaluate cellular damage induced by oxidative stress in epidermal cells, a human epidermoid carcinoma cell line in culture (A 431) was used as experimental model. Cell treatment with W-oxidized LDL resulted in cytostatic and cytotoxic effects characterized by morphological and functional alterations: inhibition of cell proliferation, modifications of cytoskeleton network, microtubular derangement, loss of cell-cell and cell-substrate contacts, cell detachment and cell death by apoptosis, The ox-LDL-induced alterations were almost completely prevented by pre-incubating cells with alpha-tocopherol. The results presented here could be of relevance for a better comprehension of the pathogenic mechanisms of several human diseases, including dermatological pathologies, and could indicate that antioxidants such as ar-tocopherol could represent an important therapeutic challenge in the maintenance of cell and tissue homeostasis in the long run.

关键词：上皮细胞/药物作用上皮细胞/代谢上皮细胞/超微结构脂蛋白类 LDL/生理学脂蛋白类 LDL/辐射效应显微镜检查电子扫描氧化性应激亚细胞部分/药物作用亚细胞部分/代谢肿瘤细胞培养的紫外线维生素E/药理学人类

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Flavone antagonists bind competitively with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation

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MOLECULAR PHARMACOLOGY 1999年第4期55卷 716-725页

作者： Henry, EC Kende, AS Rucci, G Totleben, MJ Willey, JJ Dertinger, SD Pollenz, RS Jones, JP Gasiewicz, TA Univ Rochester Med Ctr Dept Environm Med Rochester NY 14642 USA Univ Rochester Dept Chem Rochester NY 14642 USA Univ Rochester Dept Physiol & Pharmacol Rochester NY 14642 USA Med Univ S Carolina Dept Biochem & Mol Biol Charleston SC 29425 USA

Previous analyses suggested that potent aryl hydrocarbon receptor (AhR) antagonists were planar, with a lateral electron-rich center. To further define structural requirements and mechanism for antagonism, ten additional flavone derivatives were synthesized. Based on their ability to 1) compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the AhR;2) inhibit TCDD-elicited binding of AhR to dioxin-responsive elements (DRE) in vitro;and 3) inhibit TCDD-induced transcription of DRE;dependent luciferase in stably transfected hepatoma cells, the most potent flavones contained a 3'-methoxy group and a 4'-substituent having one or more terminal atoms of high electron density (-N-3, - NO2, or -NCS). Furthermore, these had low agonist activity as assessed by their inability to elicit AhR DRE binding or to induce luciferase. Compounds containing bulkier 3' or 4'-substituents, or a 3'-OH group were less potent antagonists, and some were partial agonists. In rat liver cytosol, 3'-methoxy-4'-azido- and 3'-methoxy-4'-nitroflavones bound competitively (with TCDD) to the AhR, indicating that they bind to the TCDD-binding site. When hepatoma cells were exposed to these flavones, AhR complexes were primarily immunoprecipitable from the cytosol and contained 90 kDa heat shock protein. In contrast, AhR in TCDD-treated cells was primarily immunoprecipitated from nuclear extracts and was associated with Arnt but not 90 kDa heat shock protein. Immunocytofluorescence analysis in intact cells further indicated that the potent antagonist inhibited nuclear uptake of AhR and blocked TCDD-dependent down-regulation of AhR. Together, these data indicate that the most potent antagonists bind the AhR with high affinity but cannot initiate receptor transformation and nuclear localization.

关键词：结合部位癌肝细胞胞质溶胶/药物作用胞质溶胶/代谢减量调节类黄酮物质/药理学配体肝/药物作用肝/代谢蛋白质构象大鼠 Sprague-Dawley 受体芳基烃/拮抗剂和抑制剂受体芳基烃/化学受体芳基烃/代谢构效关系亚细胞部分/药物作用亚细胞部分/代谢四氯二苯并二恶英/药理学转录遗传/药物作用肿瘤细胞培养的动物男(雄)性小鼠大鼠

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Antagonism of immunostimulatory CpG-oligodeoxynucleotides by 4-aminoquinolines and other weak bases: Mechanistic studies

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JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 1999年第3期291卷 1337-1347页

作者： Manzel, L Strekowski, L Ismail, FMD Smith, JC Macfarlane, DE Univ Iowa Dept Med Iowa City IA 52242 USA Vet Affairs Med Ctr Dept Med Iowa City IA 52242 USA

Oligodeoxynucleotides with unmethylated CpG motifs are immunostimulatory. Chloroquine and a number of structural analogs specifically and powerfully inhibit this effect at nanomolar concentrations. We explored the mechanism of this inhibition, with 4-aminoquinolines, quinacrine, 9-aminoacridines, and novel dibasic analogs, many of which are fluorescent. WEHI 231 murine B-lymphoma cells accumulated analogs up to a concentration several hundredfold higher than the medium. Uptake was rapid, nonsaturable, reversible, and partially inhibited by monensin, an agent that collapses pH gradients within cells. Uptake did not correlate highly with efficacy as inhibitors of CpG-oligodeoxynucleotide (ODN)-induced effects, suggesting that analogs act by a specific action. Confocal microscopy revealed analogs concentrating in large peripheral organelles. CpG-ODN is taken up by cells into acidifed, small, perinuclear vesicles. This uptake is thought to be necessary for immunostimulatory activity. Cellular uptake of fluorescent CpG-ODN was not inhibited by the analogs. The pH of intracellular CpG-ODN (6.4) was not affected by analogs at the concentration required for inhibition, but pH was increased by higher concentrations. UV spectroscopy revealed no binding of analogs to CpG-ODN. Nuclear Overhauser effect spectroscopy revealed that an analog bound to phosphatidylcholine vesicles, with the ring structure of the analog buried within the lipid and the side chain facing the aqueous environment. We conclude that the analogs do not inhibit the action of CpG-ODN by preventing the uptake or acidification of CpG-ODN. It seems more likely that the analogs inhibit the efficacy of CpG-ODN by a specific action within acidified vesicles, possibly at the interface of a phospholipid membrane.

关键词：佐剂免疫/拮抗剂和抑制剂佐剂免疫/药理学碱类/药理学氨基喹啉类/药理学 CpG岛 DNA 单链/药物作用氢离子浓度离子载体/药理学脂类/化学磁共振波谱学膜人工显微镜检查共焦莫能星/药理学寡核苷酸类/拮抗剂和抑制剂寡核苷酸类/代谢寡核苷酸类/药理学磷脂类/化学亚细胞部分/药物作用亚细胞部分/代谢肿瘤细胞培养的

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TEMPERATURE-DEPENDENT INHIBITION OF POLYPHENYLALANINE FORMATION BY EXOTOXIN OF BACILLUS-THURINGIENSIS

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FEBS LETTERS 1975年第3期54卷 330-333页

作者： SOMERVILLE, HJ SWAIN, HM SHELL RES LTD WOODSTOCK LABRES CTRSITTINGBOURNE ME9 8AGKENTENGLAND

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Translocation of TRAF proteins regulates apoptotic threshold of cells

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第3期272卷 936-945页

作者： Arch, RH Gedrich, RW Thompson, CB Univ Chicago Gwen Knapp Ctr Lupus & Immunol Res Chicago IL 60637 USA Univ Chicago Howard Hughes Med Inst Chicago IL 60637 USA Univ Chicago Dept Med Chicago IL 60637 USA

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are involved in signaling pathways triggered by members of the TNF receptor (TNFR) family and other cell surface proteins. After recruitment to a receptor, TRAFs initiate formation of multiprotein complexes that induce downstream events, such as translocation of transcription factor nuclear factor kappa B (NF-kappa B) and activation of c-Sun N-terminal kinase (JNK). Several proteins in these complexes play important roles in regulation of apoptosis. However, the fate of TRAF-containing complexes once assembled in response to receptor multimerization is not understood. In this report, we demonstrate that crosslinking of TNFR family members or interaction of TRAF2 with the cytoplasmic protein A20 leads to intracellular translocation of TRAF2. This redistribution leads to depletion of the cytoplasmic pool of TRAF2. The ratio between soluble and insoluble TRAF2 determines the sensitivity of cells to TNF-alpha-induced apoptosis and may play an important role in limiting further TRAF-dependent signal transduction. (C) 2000 Academic Press.

关键词：抗原 CD 抗原 CD137 抗原 CD27/遗传学抗原 CD27/代谢抗原 CD30/化学抗原 CD30/遗传学抗原 CD30/代谢细胞凋亡/药物作用结合部位生物转运/药物作用细胞系细胞质/药物作用细胞质/代谢脱氧核糖核酸酶Ⅰ/代谢药物耐受性内质网/药物作用内质网/代谢细胞内信号肽和蛋白质类 NF-κB/代谢核蛋白质类容积渗克分子浓度蛋白质结合/药物作用蛋白质类/遗传学蛋白质类/代谢受体神经生长因子类/遗传学受体神经生长因子类/代谢受体 OX40 受体肿瘤坏死因子/遗传学受体肿瘤坏死因子/代谢重组融合蛋白质类/遗传学重组融合蛋白质类/代谢信号传导/药物作用溶解度亚细胞部分/药物作用亚细胞部分/代谢 TNF受体相关因子2 转染肿瘤坏死因子α/药理学人类

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DNA-SYNTHESIS INVOLVING A COMPLEXED FORM OF DNA-POLYMERASE I IN EXTRACTS OF ESCHERICHIA-COLI

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1975年第6期72卷 2099-2103页

作者： HENDLER, RW PEREIRA, M SCHARFF, R NHLI SECT MEMBRANE ENZYMOLLAB CELL BIOLBETHESDAMD 20014

DNA polymerase I (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) has been recovered as a complex of about 390,000 molecular weight. The complex displays an ATP-stimulated DNA-synthesizing activity that prefers native to heat-denatured DNA. Genetic evidence indicates that the recBC enzyme is associated with the polymerase in the complex. Preliminary evidence for complexes involving DNA polymerases II and III is also presented.

关键词：腺苷三磷酸/药理学色谱法凝胶 DNA核苷酸转移酶类/分离和提纯 DNA 细菌/药理学电泳聚丙烯酰氨凝胶酶激活大肠杆菌/药物作用大肠杆菌/酶学大肠杆菌/超微结构遗传学微生物分子量突变核酸变性作用氯化钾/药理学亚细胞部分/药物作用亚细胞部分/酶学

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KINETICS OF OPIATE RECEPTOR INACTIVATION BY SULFHYDRYL REAGENTS - EVIDENCE FOR CONFORMATIONAL CHANGE IN PRESENCE OF SODIUM IONS

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1975年第6期72卷 2404-2407页

作者： SIMON, EJ GROTH, J NYU MED CTRDEPT MEDNEW YORKNY 10016

The role of SH groups in opiate-receptor interactions has been further examined. In activation by N-ethylmaleimide of sterospecific opiate binding by rat brain membrane fractions follows pseudo-first order kinetics and exhibits strong temperature dependence. The kinetics indicate that alkylation of a single SH group suffices to block opiate binding. Considerable protection from SH group inactivation is observed when treatment with N-ethylmaleimide is carried out in the presence of an opiate or an antagonist, suggesting close proximity of the SH group to the opiate binding site. The rate of inactivation of receptor binding by N-ethylmaleimide is markedly slower in buffers containing 100 mM NaCl (t1/2 equals 30 plus or minus 1.4 min) than in sodium-free buffers (t1/2 equals 10 plus or minus 1.0 min). Since the rate of alkylation of model SH compounds is unaffected by sodium ions, this protection seems best explained by a conformational change in the receptors that renders the SH groups less accessible to alkylation. The rate of inactivation is not affected by K+, Rb+, or Cs+ and only slightly by Li+. This cation specificity as well as the concentration-response to Na+ are remarkably similar to those previously shown to lead to increased antagonist and decreased agonist binding. We suggest that the same conformational change is involved in the two phenomena.

关键词：烷化作用结合部位脑/药物作用脑/代谢脑/超微结构环丙烷类/代谢乙基马来酰亚胺/药理学埃托啡/代谢动力学纳洛酮/类似物和衍生物纳洛酮/代谢麻醉品/代谢蛋白质构象受体药物氯化钠/药理学亚细胞部分/药物作用亚细胞部分/代谢巯基试剂/药理学温度动物大鼠

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