Journal of receptor research最新文献_第6页

A method for radioligand binding assays using a robotic workstation. 一种使用机器人工作站的放射配体结合测定方法。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073650

B Cusack, E Richelson

{"title":"A method for radioligand binding assays using a robotic workstation.","authors":"B Cusack, E Richelson","doi":"10.3109/10799899309073650","DOIUrl":"https://doi.org/10.3109/10799899309073650","url":null,"abstract":"Radioligand binding assays provide a powerful tool for screening drug candidates at many receptors. We present a method utilizing a robotic workstation, the Biomek 1000, which automates the tedious and repetitive tasks of these assays. First, the robot handles the serial dilution of up to 8 drugs with 11 concentrations per drug. The sequential addition of diluting buffer, non-radioactively labeled ligand, radioactive ligand, and finally the tissue homogenate or membrane preparation, is fully automated. A novel rack design allows the use of tubes with a maximum capacity of 2 ml, providing a total assay volume of 1 ml. Final filtration on a Brandel cell harvester outfitted with a uniquely designed head allows for processing of 48 samples simultaneously from the rack holder. We have employed this method for the determination of equilibrium dissociation constants (Kds) for drugs at the 5 human muscarinic acetylcholine receptor subtypes expressed in cultured cells, as well as histamine H1, dopamine D2, serotonin 5HT1A, alpha 1- and alpha 2-adrenergic receptors in human brain tissue homogenates. Our results compare favorably with manual methods reported for these receptors, and exhibit a very high degree of reproducibility and throughput, with a minimum of operator input.","PeriodicalId":16948,"journal":{"name":"Journal of receptor research","volume":"13 1-4","pages":"123-34"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/10799899309073650","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19433777","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}

引用次数: 17

Receptor binding and biological activity of IL-1 alpha, IL-1 beta, IL-1 beta analogues and an IL-1 antagonist in A375 human melanoma cells. IL-1α、IL-1β、IL-1α类似物和IL-1拮抗剂在A375人黑色素瘤细胞中的受体结合和生物活性。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073658

J B Baumann, E Christen, G Gamboni, U Joss, J van Oostrum, J Girard, A N Eberle

{"title":"Receptor binding and biological activity of IL-1 alpha, IL-1 beta, IL-1 beta analogues and an IL-1 antagonist in A375 human melanoma cells.","authors":"J B Baumann, E Christen, G Gamboni, U Joss, J van Oostrum, J Girard, A N Eberle","doi":"10.3109/10799899309073658","DOIUrl":"https://doi.org/10.3109/10799899309073658","url":null,"abstract":"A receptor binding assay for IL-1 peptides on human melanoma cells of the A 375 cell line is reported. Strains differing in their sensitivity to the cytotoxic effects of IL-1 beta were compared. In both strains, binding equilibrium at temperatures between 0 degrees and 37 degrees C was reached after 4 to 8 hours. At 37 degrees C, most of the bound ligand was rapidly internalized leaving a constant level of surface receptors. Scatchard analysis at 0 degrees C revealed a single class of high affinity receptors with a similar KD in both IL-1 resistant (0.18 +/- 0.07 nM) and sensitive strains (0.14 +/- 0.06 nM) but a 10-fold difference in the number of binding sites. Whereas > 1000 binding sites per cell were regularly observed in all resistant strains, only 100-200 sites could be detected on the IL-1 sensitive cells. In displacement assays, IL-1 beta was found to be slightly more potent than IL-1 alpha in both strains. In an attempt to further characterize the IL-1 binding site in these cells, the binding characteristics and biological activity of 20 point mutations of IL-1 beta were examined. EC50 values similar to those of the wild type peptide were found in all these analogues with the exception R11S and E128K: their EC50 was increased by a factor of 10 but the biological activity was reduced 1000-fold as compared to IL-1 beta. The relative potency of an IL-1 receptor antagonist was similar to that of IL-1 beta in the displacement binding assay but a 100-fold higher concentration was required to completely block the cytotoxic effects of IL-1 beta. These results show that A375 human melanoma cells are useful for screening the binding and biological properties of analogues of the IL-1 family of peptides.","PeriodicalId":16948,"journal":{"name":"Journal of receptor research","volume":"13 1-4","pages":"245-62"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/10799899309073658","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19433779","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}

引用次数: 4

Design, synthesis and some uses of receptor-specific agonists and antagonists of vasopressin and oxytocin. 抗利尿激素和催产素受体特异性激动剂和拮抗剂的设计、合成和一些用途。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073655

M Manning, W H Sawyer

引用次数: 86

Peptides containing multiple photolabels: a new tool for the analysis of ligand-receptor interactions. Reversible long-lasting stimulation and inhibition of MSH receptors by multiple photocrosslinks with alpha-MSH. 含有多个光标记的肽:一种分析配体-受体相互作用的新工具。与-MSH的多重光交联对MSH受体的可逆持久刺激和抑制。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073643

A N Eberle

{"title":"Peptides containing multiple photolabels: a new tool for the analysis of ligand-receptor interactions. Reversible long-lasting stimulation and inhibition of MSH receptors by multiple photocrosslinks with alpha-MSH.","authors":"A N Eberle","doi":"10.3109/10799899309073643","DOIUrl":"https://doi.org/10.3109/10799899309073643","url":null,"abstract":"Crosslinking of MSH receptors on melanophores of the lizard Anolis carolinensis with analogues of alpha-MSH containing a photoreactive group in position 1, 7, 9 or 13 leads to long-lasting receptor stimulation. Reversibility of this long-lasting stimulation is obtained by employing a disulfide-containing photoreactive group which can be cleaved from the receptor by thiol reagents [Ref. 3]. When two photoreactive groups are simultaneously present on the alpha-MSH molecule (e.g in positions 1 + 9; 1 + 13; 7 + 13, or 9 + 13), identical results were obtained and long-lasting receptor stimulation was not altered after cleavage of one single crosslink. alpha-MSH analogues with three photoreactive groups in positions 1 + 7 + 13 led to irreversible receptor stimulation whereas one compound with the photoreactive groups in positions 1 + 9 + 13 induced reversible receptor inactivation which could be changed into long-lasting stimulation by cleaving the crosslink at position 1 of alpha-MSH. These results demonstrate that one and the same peptide ligand may contain structural information for both receptor activation and inhibition and that the receptor may become arrested in an activated or inhibited state by multiple photocrosslinking, depending on the relative positions of these crosslinks.","PeriodicalId":16948,"journal":{"name":"Journal of receptor research","volume":"13 1-4","pages":"27-37"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/10799899309073643","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19368607","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}

引用次数: 4

Biochemical characterization and solubilization of human NK2 receptor expressed in Chinese hamster ovary cells. 人NK2受体在中国仓鼠卵巢细胞表达的生化表征及增溶作用。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073684

G Turcatti, K Ceszkowski, A Chollet

引用次数: 10

Transcription activation by nuclear receptors. 核受体的转录激活。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073686

H Gronemeyer

{"title":"Transcription activation by nuclear receptors.","authors":"H Gronemeyer","doi":"10.3109/10799899309073686","DOIUrl":"https://doi.org/10.3109/10799899309073686","url":null,"abstract":"Nuclear receptors constitute a superfamily of ligand-inducible transcription factors which respond to endocrine, paracrine and, possibly, autocrine signals. Multiple regulatory mechanisms assure that signal transduction results in an accurate regulation of the respective gene networks. Apart from selective expression of the cognate receptor and its binding to specific hormone response elements of target genes, additional mechanisms are responsible for the cell- and promoter-specific transcription activation. They are based on the \"interpretation\" of the signal by the multiple functional modules of a given receptor and involve a specific interplay with various factors binding to complex target gene promoters and cell-specific intermediary transcription factors that mediate the activity of the two receptor transcription activation functions, as well as homo- and heterodimerization, and interference with other signalling pathways. Moreover, a single ligand may initiate different gene programs due to the differential target gene specificities of nuclear receptor isoforms. Thus, signal transduction by nuclear receptors involves a multitude of interactive elements, as could have been expected from the central role of these signals in homeostasis, embryonic development and differentiation. Two distinct mechanisms are involved in anti-hormone action. Type I anti-hormones impair the activity of the transcription activation function, while type II antagonists impair DNA binding. Experiments aimed at an understanding of these mechanisms are discussed.","PeriodicalId":16948,"journal":{"name":"Journal of receptor research","volume":"13 1-4","pages":"667-91"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/10799899309073686","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19369692","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}

引用次数: 25

Targeting of the Gi2 alpha gene in ES cells with replacement and insertion vectors. 用替代和插入载体靶向胚胎干细胞中的Gi2 α基因。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073683

U Rudolph, P Brabet, J Kaplan, P Hasty, A Bradley, L Birnbaumer

引用次数: 6

Specific CSF-1 binding on murine placental trophoblasts and macrophages serves as a link to placental growth. 小鼠胎盘滋养细胞和巨噬细胞的特异性CSF-1结合与胎盘生长有关。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073690

I Athanassakis-Vassiliadis, J Papamatheakis, S Vassiliadis

{"title":"Specific CSF-1 binding on murine placental trophoblasts and macrophages serves as a link to placental growth.","authors":"I Athanassakis-Vassiliadis, J Papamatheakis, S Vassiliadis","doi":"10.3109/10799899309073690","DOIUrl":"https://doi.org/10.3109/10799899309073690","url":null,"abstract":"Previous studies have shown that the colony-stimulating factor-1 (CSF-1), stimulates the in vitro proliferation of a fetally-derived adherent, phagocytic and non-specific esterase positive placental cell population which stains positively for cytokeratin and Mac-1. Binding experiments were designed to test whether this is a direct effect of the factor on these cells. Binding/elution as well as autoradiography experiments, show that adherent placental cells specifically bind CSF-1. Based on the expression of the endothelial markers cytokeratin and vimentin three subpopulations of cells were isolated from the murine placenta: labyrinthine-derived trophoblasts (cytokeratin positive, vimentin negative), spongiotrophoblast-derived trophoblasts (cytokeratin positive, vimentin negative) and placental macrophages (cytokeratin negative, vimentin positive). 3H-Thymidine incorporation assays as well as binding experiments, showed that these cells simultaneously respond to and bind the macrophage-specific factor CSF-1. Furthermore, the results indicate that isolated trophoblasts have a low rate of growth and they are very sensitive to mitogenic stimulation, whereas placental macrophages alone have a high rate of growth and therefore are less sensitive to the mitogenic stimulus. These findings are in favour of the existence of an important cytokine regulatory network in the murine placenta, where two major cell populations may collaborate possibly via soluble factors to stimulate placental growth and thus fetal development.","PeriodicalId":16948,"journal":{"name":"Journal of receptor research","volume":"13 1-4","pages":"739-51"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/10799899309073690","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19434355","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}

引用次数: 16

Muscarinic receptor regulation and 2nd messenger responses in rat neocortex cultures. 大鼠新皮层毒蕈碱受体调控和第二信使反应。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073671

F van Huizen, J A Tonnaer

{"title":"Muscarinic receptor regulation and 2nd messenger responses in rat neocortex cultures.","authors":"F van Huizen, J A Tonnaer","doi":"10.3109/10799899309073671","DOIUrl":"https://doi.org/10.3109/10799899309073671","url":null,"abstract":"Primary cultures of dissociated cerebral cortex cells were used to characterize the muscarinic acetylcholinergic receptors (mAChR) present and to study receptor down-regulation and receptor mediated 2nd messenger responses induced by muscarinic agonists. Binding of the hydrophilic antagonist [3H]N-methyl scopolamine ([3H]NMS) to the cultured cells was saturated after one hour at 4 degrees C with a Kd of 93 pM and a Bmax of 958 fmol/mg protein. Competition binding studies with several antagonists and agonists indicated that the mAChR present in the culture were of a mixed M1/M3 subtype. The number of muscarinic receptors at the cell surface decreased by 60% after one hour pre-incubation of the cultures with 10 microM carbachol or oxotremorine. After down-regulation with carbachol affinity for pirenzepine was decreased, while low affinity sites for 4-DAMP were lost, indicating that especially M1 subtypes are sensitive to this type of regulation. Carbachol and oxotremorine-M induced a 2-3 fold increase in phosphatidyl inositide (PI) turnover, which was blocked with high affinity by both pirenzepine and 4-DAMP. Down-regulation of the mAChR and stimulation of PI-turnover by agonists with different potency and intrinsic activity appeared highly correlated. These data suggest that activation of the PI second-messenger system is involved in the desensitization and down-regulation of the muscarinic acetylcholine receptor.","PeriodicalId":16948,"journal":{"name":"Journal of receptor research","volume":"13 1-4","pages":"437-51"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/10799899309073671","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19434379","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}

引用次数: 5

Errors introduced in radioligand binding studies due to displaceable, cation dependent, [3H]prazosin binding to glass-fibre filters and glass surfaces. 放射性配体结合研究中由于可置换、阳离子依赖、[3H]吡唑嗪与玻璃纤维过滤器和玻璃表面的结合而引入的错误。

Journal of receptor research Pub Date : 1993-01-01 DOI: 10.3109/10799899309073694

D M Veenstra, K J van Buuren, M J Krielaart, F P Nijkamp

{"title":"Errors introduced in radioligand binding studies due to displaceable, cation dependent, [3H]prazosin binding to glass-fibre filters and glass surfaces.","authors":"D M Veenstra, K J van Buuren, M J Krielaart, F P Nijkamp","doi":"10.3109/10799899309073694","DOIUrl":"https://doi.org/10.3109/10799899309073694","url":null,"abstract":"[3H]prazosin not only specifically and homogeneously labels alpha 1-adrenoceptors, but also binds to glass surfaces and non-linearly to the glass-fibre filters, commonly used in radioligand binding experiments. Binding to filters can be modulated by unlabeled alpha-adrenergic compounds and cations. If no correction is applied for displaceable filter binding, analysis of [3H]prazosin binding experiments leads to erroneous results. Analysis of [3H]prazosin saturation experiments on guinea-pig cerebral cortex membranes with correction for filter binding before the non-linear fit procedure indicated that [3H]prazosin labels a homogeneous population of alpha 1-adrenoceptors (Rtot: 8.33 fmol.mg-1 wet tissue) with a dissociation constant of 1.28 x 10(-10) M. However, analysis of the same data after correction for non-specific binding, (determined in parallel experiments by adding 10 microM phentolamine to the incubation medium) resulted in a best fit to a model in which [3H]prazosin labels two alpha 1-adrenoceptor subpopulations (R1: 15.0 fmol.mg-1 and R2: 14.6 fmol.mg-1 wet tissue) with dissociation constants of respectively 1.78 x 10(-10) and 5.63 x 10(-9) M. The discrepancy between the two methods of analysis is due to displacement of the radioligand from the filters by phentolamine. Prazosin and oxymetazoline are also able to displace filter-bound [3H]prazosin. The extent to which displaceable filter binding distorts the proper results depends on the actual magnitude of the error and also on the method of analysis.","PeriodicalId":16948,"journal":{"name":"Journal of receptor research","volume":"13 5","pages":"801-14"},"PeriodicalIF":0.0,"publicationDate":"1993-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3109/10799899309073694","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19445970","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}

引用次数: 5