scholarly journals Cytokinin activity ofN6-benzyladenine derivatives assayed by interaction with the receptorsin planta, in vitro, andin silico

2018 ◽  
Author(s):  
Ekaterina M. Savelieva ◽  
Vladimir E. Oslovsky ◽  
Dmitry S. Karlov ◽  
Nikolay N. Kurochkin ◽  
Irina A. Getman ◽  
...  
Keyword(s):  
Specific Binding ◽  
Biological Effects ◽  
Test System ◽  
Membrane Receptors ◽  
Receptor Interaction ◽  
Cytokinin Activity ◽  
Interaction Patterns ◽  
In Planta ◽  
Cytokinin Receptors

AbstractBiological effects of hormones in both plants and animals are based on high-affrnity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived inArabidopsisby three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin–receptor interaction, we used 25 derivatives of potent cytokininN6-benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors fromArabidopsis. The mainin plantaassay system was based onArabidopsisdouble mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest withAmaranthusseedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in thein vitrotest system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, replacement ofN6by sulfur or oxygen, resulted in suppression of cytokinin activity of the derivative towards all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors inArabidopsisandAmaranthuswere revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between extent of compound binding to a receptorin vitroand its ability to activate the same receptorin plantawere evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells.Graphical abstractIndividual cytokinin receptors fromArabidopsiswere assayedin planta,in vitroandin silicowith 25 different 6-benzyladenine derivatives, new receptor-specific cytokinins were revealed.

The glucocorticoid receptor binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential mechanism for negative regulation

1991 ◽  
Vol 11 (6) ◽  
pp. 3379-3383
Author(s):  
P E Strömstedt ◽  
L Poellinger ◽  
J A Gustafsson ◽  
J Carlstedt-Duke
Keyword(s):  
Glucocorticoid Receptor ◽  
Specific Binding ◽  
Tata Box ◽  
Receptor Interaction ◽  
Receptor Binding Site ◽  
Osteocalcin Gene ◽  
Close Proximity ◽  
Human Osteocalcin

Expression of the human osteocalcin promoter is negatively regulated by glucocorticoids in vivo. In vitro DNase I and exonuclease III footprinting analysis showed binding of purified glucocorticoid receptor in close proximity to and overlapping with the TATA box of the osteocalcin gene. These results imply competition or interference with binding of the TATA box-binding transcription factor IID as a mechanism of repression of this gene by glucocorticoids. In support of this notion, point mutation analysis of the receptor binding site indicated that flanking nucleotides and not the TATA box motif per se were important for receptor interaction. Moreover, DNA binding competition assays showed specific binding of the receptor only to the TATA box region of the osteocalcin gene and not to the corresponding region of an immunoglobulin heavy-chain promoter.

scholarly journals Somatostatin and dopamine receptor interaction in prostate and lung cancer cell lines

2010 ◽  
Vol 207 (3) ◽  
pp. 309-317 ◽  
Author(s):  
M Arvigo ◽  
F Gatto ◽  
M Ruscica ◽  
P Ameri ◽  
E Dozio ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Membrane Receptors ◽  
Receptor Interaction ◽  
Inhibition Of Proliferation ◽  
Receptor Interactions

Somatostatin analogues inhibit in vitro cell proliferation via specific membrane receptors (SSTRs). Recent studies on transfected cell lines have shown a ligand-induced formation of receptor dimers. The aim of this study is 1) to evaluate the role of specific ligands in modulating receptor interactions in the androgen-dependent prostate cancer cell line, LNCaP, and in the non-small cell lung cancer line, Calu-6, by co-immunoprecipitation and immunoblot; and 2) to correlate the antiproliferative effect of these compounds with their ability in modulating receptor interactions. In LNCaP, we have demonstrated the constitutive presence of sstr1/sstr2, sstr2/sstr5, sstr5/dopamine (DA) type 2 receptor (D2R), and sstr2/D2R dimers. BIM-23704 (sstr1- and sstr2-preferential compound) increased the co-immunoprecipitation of sstr1/sstr2 and significantly inhibited proliferation (−30.98%). BIM-23244 (sstr2–sstr5 selective agonist) significantly increased the co-immunoprecipitation of sstr2/sstr5, and induced a −41.36% inhibition of proliferation. BIM-23A760, a new somatostatin/DA chimeric agonist with a high affinity for sstr2 and D2R and a moderate affinity for sstr5, significantly increased the sstr5/D2R and sstr2/D2R complexes and was the most powerful in inhibiting proliferation (−42.30%). The chimeric compound was also the most efficient in modulating receptor interaction in Calu-6, increasing the co-immunoprecipitation of D2R/sstr5 and inhibiting cell proliferation (−30.54%). However, behind BIM-23A760, BIM-53097 (D2R-preferential compound) also significantly inhibited Calu-6 proliferation (−17.71%), suggesting a key role for D2R in receptor cross talk and in controlling cell growth. Indeed, activation of monomeric receptors did not affect receptor co-immunoprecipitation, whereas cell proliferation was significantly inhibited when the receptors were synergistically activated. In conclusion, our data show a dynamic ligand-induced somatostatin and DA receptor interaction, which may be crucial for the antiproliferative effects of the new analogues.

scholarly journals A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds

1969 ◽  
Vol 111 (4) ◽  
pp. 487-495 ◽  
Author(s):  
M K Johnson
Keyword(s):  
Organophosphorus Compounds ◽  
Specific Binding ◽  
Phosphorylation Site ◽  
Test System ◽  
Binding Activity ◽  
Specific Component ◽  
Esterase Inhibitors ◽  
Brain And Spinal Cord

1. It is proposed that part of a neurotoxic dose of di-isopropyl phosphorofluoridate will be covalently bound in vivo to a specific component in the brain and spinal cord as the initial biochemical event in the genesis of the lesion. 2. A test system in vitro was devised that removes many di-isopropyl phosphorofluoridate-binding sites and indicates that the specific component may be a protein present in brain at a concentration comparable with that of the cholinesterases. 3. The site was found to be present and capable of binding di-isopropyl phosphorofluoridate in vitro in brain samples taken from either normal hens or those dosed with organophosphorus esterase inhibitors that are not neurotoxic. 4. Very little of the specific binding activity was found in brain samples from hens pre-dosed with a variety of neurotoxic organophosphorus compounds. 5. A solubilized preparation of the active brain component was obtained, suitable for further purification and study.

Studies of cytokinin receptor–phosphotransmitter interaction provide evidences for the initiation of cytokinin signalling in the endoplasmic reticulum

2018 ◽  
Vol 45 (2) ◽  
pp. 192 ◽  
Author(s):  
Sergey N. Lomin ◽  
Yulia A. Myakushina ◽  
Dmitry V. Arkhipov ◽  
Olga G. Leonova ◽  
Vladimir I. Popenko ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Endoplasmic Reticulum ◽  
Active Form ◽  
Kinase Assay ◽  
Phosphoryl Group ◽  
Subcellular Localisation ◽  
In Planta ◽  
Cytokinin Receptors ◽  
Receptor Dimers

Cytokinin receptors were shown recently to be localised mainly to the endoplasmic reticulum (ER); however, the activity of ER-located receptors was not proven. We have therefore tested the functionality of ER-located Arabidopsis receptors. The first step of cytokinin signal transduction is the transfer of a phosphoryl group from the activated receptor to a phosphotransfer protein. To determine the subcellular localisation of receptor–phosphotransmitter interaction in planta, BiFC experiments were performed. Receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4 (CRE1) and phosphotransmitters ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTER 1 (AHP1), AHP2 and AHP3 fused to split-eYFP were transiently expressed in Nicotiana benthamiana leaves. Receptor–phosphotransmitter pairs were shown to interact in every possible combination in a pattern reflecting the ER. Receptor dimers, an active form of the receptors, were also detected in the ER. According to BiFC and protease protection data, the catalytic part of AHK3 was located in the cytoplasm whereas the hormone binding module faced the ER lumen. This topology is consistent with receptor signalling from the ER membrane. Finally, the functionality of receptors in different membrane fractions was tested using an in vitro kinase assay visualising the phosphorylation of phosphotransfer proteins. The detected cytokinin-dependent phosphotransfer activity was confined mainly to the ER-enriched fraction. Collectively, our data demonstrate that ER-located cytokinin receptors are active in cytokinin signal transduction. Hence, intracellular cytokinins appear to play an essential role in cytokinin signalling. An updated model for the spatial organisation of cytokinin transport form activation, intracellular trafficking and signalling from the ER is proposed.

IDENTIFICATION OF FOUR TYPES OF STEROID BY THEIR INTERACTION WITH MINERALOCORTICOID RECEPTORS IN THE TOAD BLADDER

1970 ◽  
Vol 48 (4) ◽  
pp. 563-574 ◽  
Author(s):  
K. G. M. M. ALBERTI ◽  
G. W. G. SHARP
Keyword(s):  
Sodium Transport ◽  
Specific Binding ◽  
Biological Response ◽  
Toad Bladder ◽  
Receptor Interaction ◽  
Mineralocorticoid Receptors ◽  
Multiple Points ◽  
Specific Binding Sites ◽  
Receptor Sites

SUMMARY The effects of various steroids on sodium transport across the toad bladder were examined in vitro. Cortisone, alone, had no effect on sodium transport but was an antagonist of aldosterone. Cortisone was shown to bind to the two non-specific binding sites for steroids in the nucleus of the toad bladder epithelial cells when present at a concentration of 10−7m. At higher concentrations it interacted with the mineralocorticoid receptors and displaced aldosterone from these sites. Cortexolone had a weak stimulating effect on sodium transport and also antagonized the effect of aldosterone. Cortexolone, when present in excess, displaced aldosterone from its receptor sites. Four types of steroid could be differentiated by their interaction with the mineralocorticoid receptor sites and the subsequent biological response: (a) steroids that bind to the receptors and produce the full biological effect on sodium transport; (b) steroids that do not bind and therefore are incapable of producing a response; (c) steroids that bind to the receptor sites, have no effect alone but inhibit the action of aldosterone; (d) steroids that bind to the receptor sites, produce a partial response on sodium transport and subsequently antagonize the action of aldosterone. Multiple points of attachment are postulated for the steroid—receptor interaction.

scholarly journals ICBP90, a Novel Methyl K9 H3 Binding Protein Linking Protein Ubiquitination with Heterochromatin Formation

2007 ◽  
Vol 28 (2) ◽  
pp. 705-717 ◽  
Author(s):  
Panagiota Karagianni ◽  
Larbi Amazit ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Specific Binding ◽  
Binding Protein ◽  
Biological Effects ◽  
Binding Activity ◽  
Effector Proteins ◽  
Heterochromatin Formation ◽  
Protein Ubiquitination

ABSTRACT Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells.

BINDING AND METABOLISM OF 5α-ANDROSTANE-3α,17β-DIOL AND OF 5α-ANDROSTANE-3β,17β-DIOL IN THE PROSTATE, SEMINAL VESICLES AND PLASMA OF MALE RATS: STUDIES IN VIVO AND IN VITRO

1975 ◽  
Vol 64 (3) ◽  
pp. 529-538 ◽  
Author(s):  
M. KRIEG ◽  
H.-J. HORST ◽  
M.-L. STERBA
Keyword(s):  
Skeletal Muscle ◽  
Specific Binding ◽  
Biological Effects ◽  
Sucrose Density Gradient ◽  
Total Radioactivity ◽  
Seminal Vesicles ◽  
Male Rats ◽  
Cytosol Fraction

SUMMARY Binding of 5α-androstane-3α,17β-diol (3α-diol) and 5α-androstane-3β,17β-diol (3β-diol) in vivo and in vitro to the 100000 g cytosol fraction of the rat prostate and seminal vesicles as well as to plasma was studied by agargel electrophoresis and sucrose density gradient ultracentrifugation and the results compared with the corresponding findings for 5α-dihydrotestosterone (5α-DHT). The metabolism of 3α-diol and 3β-diol was also investigated by thin-layer chromatography. The following results were obtained: (1) A specific binding of 3α-diol and 3β-diol by the cytosols could not be demonstrated in vitro, while 5α-DHT was specifically bound. (2) In plasma, 3α-diol was extensively bound, 3β-diol less extensively bound, while 5α-DHT remained unbound. (3) After intravenous injection of 3α-diol, specifically bound radioactivity, increasing within 30 min, was found in the prostate cytosol, while after 3β-diol injection no binding occurred. (4) Parallel to the increased binding, the total radioactivity in the prostate accumulated within 30 min after 3α-diol injection, the uptake being 5·3 times higher than in skeletal muscle. However after 3β-diol injection, total radioactivity decreased in the prostate within 30 min, the uptake being only 1·5 times higher than in skeletal muscle. (5) One minute after injection of 3α-diol, 53% of the extracted radioactivity in the prostate had been converted to 5α-DHT, this increased within 30 min to 81%. Thirty minutes after the injection of 3β-diol, about 32% of the extracted radioactivity in the prostate had been converted to 5α-DHT. (6) From the in-vivo and in-vitro experiments it was concluded that 3α-diol exerts its biological effects mainly by its conversion into 5α-DHT.

scholarly journals The glucocorticoid receptor binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential mechanism for negative regulation.

1991 ◽  
Vol 11 (6) ◽  
pp. 3379-3383 ◽  
Author(s):  
P E Strömstedt ◽  
L Poellinger ◽  
J A Gustafsson ◽  
J Carlstedt-Duke
Keyword(s):  
Glucocorticoid Receptor ◽  
Specific Binding ◽  
Tata Box ◽  
Receptor Interaction ◽  
Receptor Binding Site ◽  
Osteocalcin Gene ◽  
Close Proximity ◽  
Human Osteocalcin

Expression of the human osteocalcin promoter is negatively regulated by glucocorticoids in vivo. In vitro DNase I and exonuclease III footprinting analysis showed binding of purified glucocorticoid receptor in close proximity to and overlapping with the TATA box of the osteocalcin gene. These results imply competition or interference with binding of the TATA box-binding transcription factor IID as a mechanism of repression of this gene by glucocorticoids. In support of this notion, point mutation analysis of the receptor binding site indicated that flanking nucleotides and not the TATA box motif per se were important for receptor interaction. Moreover, DNA binding competition assays showed specific binding of the receptor only to the TATA box region of the osteocalcin gene and not to the corresponding region of an immunoglobulin heavy-chain promoter.

On the Release of the Three Locust (Locusta migratoria) Adipokinetic Hormones: Effect of Crustacean Cardioactive Peptide and Inhibition by Sugars

1999 ◽  
Vol 54 (1-2) ◽  
pp. 110-118 ◽  
Author(s):  
James E. Flanigan ◽  
Gerd Gäde
Keyword(s):  
Biological Effects ◽  
Locusta Migratoria ◽  
Inhibitory Effect ◽  
Test System ◽  
Hormone Release ◽  
Adipokinetic Hormone ◽  
High Potassium ◽  
Crustacean Cardioactive Peptide

An existing test to monitor the rate of adipokinetic hormone release from the corpora cardiaca (C C) of Locusta migratoria in vitro was improved, so that a constant basal rate of release was achieved and the amount of released Lom-AKH-I, II and III could be quantified by HPLC . This test system was subsequently used to demonstrate that a small peptide, which has been found in a few insect species including L. migratoria, crustacean cardioactive peptide (CCAP), induces release of all three AKHs. Moreover, 80 mᴍ trehalose reduces CCAP-induced release of AKHs in vitro, and 160 mᴍ glucose reduces this release even further. Glucose also had a greater inhibitory effect than trehalose on the spontaneous release and inhibited the high potassium-stimulated release of AKH from the CC in vitro. Eighty mᴍ sucrose, on the other hand, had no effect on the release of AKH . The effect of trehalose and glucose could be due to their use as an energy source, with trehalose first having to be converted to glucose. Whatever the stimulus, the three AKHs are released in the same proportions as they are found in the CC, which in vivo would make Lom-AKH-I, the most abundant AKH, the major effector of the biological effects of AKHs in adult locusts

scholarly journals Three-Dimensional In Vitro Reaggregates of Embryonic Cardiomyocytes: A Potential Model System for Monitoring Effects of Bioactive Agents

2005 ◽  
Vol 10 (8) ◽  
pp. 814-822 ◽  
Author(s):  
P. Bartholomä ◽  
E. Gorjup ◽  
D. Monz ◽  
A. Reininger-Mack ◽  
H. Thielecke ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Connexin 43 ◽  
Single Cells ◽  
Cell Types ◽  
Test System ◽  
Model Systems ◽  
Receptor Interaction ◽  
Functional Markers

To understand the physiological effects of substances used in drugs and therapies on heartmuscle tissue, model systems that mirror the in vivo situation of living tissues are required. Therefore, the creation of 3-dimensional (3D) cell aggregates provides an improved and refined in vitromodel as a link between cell-free or single cells and organs orwhole organisms in vivo. Here we have characterized a stable contracting in vitro tissuemodel, which consists of embryonic chicken cardiomyocytes. For establishing a cell-based test system, the 3D in vitro cardiomyocyte spheres were characterized according to messenger RNA expression of special cardiac cell types and protein expression pattern of functional markers such as connexin-43. Finally, the in vitro spheroid modelwas used for investigating the effect of isoproterenol, a •-adrenergic receptor agonist, on the contractibility mediated by the ligand receptor interaction.

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