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81.
Santa Veiksina 《Analytical biochemistry》2010,402(1):32-39
Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC4) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligands, Cy3B-NDP-α-MSH and TAMRA-NDP-α-MSH, were used and compared in assays that monitored their binding to MC4 receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC4 receptors was saturable and with high affinity. All studied MC4 receptor-specific nonlabeled ligands displaced fluoroligands’ binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3 h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters. 相似文献
82.
B.?McCarthy A.?Daws D.?L.?MacmillanEmail author 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》2004,190(4):291-299
Recordings were made from the nerve innervating the stretch receptors of the abdominal muscle receptor organs and slow extensor muscles of tethered crayfish, Cherax destructor, during so-called non-giant swimming. The stretch receptors were active during the flexor phase of swimming but the duration and pattern of activity varied from cycle to cycle. Their pattern of firing was modified by the activity of the large accessory neurons which make direct inhibitory synapses upon them. Neither the stretch receptors nor the accessory neurons were active during the extensor phase of the cycle. The timing and extent of tailfan movements during the period of stretch receptor activity were measured from video records before and after the stretch receptor nerves were cut in the second to fifth segments. The promotion of the tailfan during flexion was significantly delayed and the minimum angle to which the uropods were remoted at the end of flexion significantly larger in denervated animals. We propose that afferent information from the stretch receptors coordinates the timing and extent of tailfan movements according to variations in the positioning and movement of the abdominal segments such that the hydrodynamic efficiency of the tailfan is enhanced on a cycle by cycle basis during non-giant swimming.Abbreviations A# abdominal segment number - Acc accessory neuron - LUU large unidentified unit - MRO muscle receptor organ - NGS non-giant swimming - SEMN slow extensor motor neuron - SR stretch receptor neuron 相似文献
83.
Prostate carcinomas are among the most frequently diagnosed and death causing cancers affecting males in the developed world. It has become clear that the molecular mechanisms that drive the differentiation of normal prostate cells towards neoplasia involve multiple signal transduction cascades that often overlap and interact. A critical mediator of cellular proliferation and differentiation in various cells (and cancers) is the cAMP-dependent protein kinase, also known as protein kinase A (PKA), and its activating secondary messenger, cAMP. PKA and cAMP have been shown to play critical roles in prostate carcinogenesis and are the subject of this review. In particular we will focus on the cross-talk between PKA/cAMP signaling and that of the androgen receptor. 相似文献
84.
Gustavo Bonaventure 《Plant signaling & behavior》2011,6(12):2060-2063
The Nicotiana attenuata LECTIN RECEPTOR KINASE 1 (LecRK1) has been recently identified as a component of the mechanism used by plants to suppress the Manduca sexta-triggered accumulation of salicylic acid (SA). The suppression of the SA burst by LecRK1 allows for the unfettered induction of jasmonic acid (JA)-mediated defense responses against M. sexta herbivory. LecRK1 contains a multi-domain extracellular region composed of a G-type Lectin domain and a PAN-AP domain separated by a variable sequence with low similarity to an EGF domain. The LecRK1 intracellular region is composed of a single domain structure with predicted Ser/Thr protein kinase activity. The multi-domain structure of the extracellular region of LecRK1 adds a level of complexity in terms of the potential ligands that this receptor protein could recognize. 相似文献
85.
Tsuyoshi Kenri Yoshito Kawakita Hisashi Kudo U. Matsumoto Shigetarou Mori Yukio Furukawa Yuhei O. Tahara Keigo Shibayama Yuuki Hayashi Munehito Arai Makoto Miyata 《Biochemical and biophysical research communications》2019,508(4):1050-1055
Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications. 相似文献
86.
Increasing number of publications shows that cannabinoid receptor 1 (CB(1)) specific compounds might act in a CB(1) independent manner, including rimonabant, a potent CB(1) receptor antagonist. Opioids, cannabinoids and their receptors are well known for their overlapping pharmacological properties. We have previously reported a prominent decrease in μ-opioid receptor (MOR) activity when animals were acutely treated with the putative endocannabinoid noladin ether (NE). In this study, we clarified whether the decreased MOR activation caused by NE could be reversed by rimonabant in CB(1) receptor deficient mice. In functional [(35)S]GTPγS binding assays, we have elucidated that 0.1mg/kg of intraperitoneal (i.p.) rimonabant treatment prior to that of NE treatment caused further attenuation on the maximal stimulation of Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO), which is a highly specific MOR agonist. Similar inhibitory effects were observed when rimonabant was injected i.p. alone and when it was directly applied to forebrain membranes. These findings are cannabinoid receptor independent as rimonabant caused inhibition in both CB(1) single knockout and CB(1)/CB(2) double knockout mice. In radioligand competition binding assays we highlighted that rimonabant fails to displace effectively [(3)H]DAMGO from MOR in low concentrations and is highly unspecific on the receptor at high concentrations in CB(1) knockout forebrain and in their wild-type controls. Surprisingly, docking computational studies showed a favorable binding position of rimonabant to the inactive conformational state of MOR, indicating that rimonabant might behave as an antagonist at MOR. These findings were confirmed by radioligand competition binding assays in Chinese hamster ovary cells stably transfected with MOR, where a higher affinity binding site was measured in the displacement of the tritiated opioid receptor antagonist naloxone. However, based on our in vivo data we suggest that other, yet unidentified mechanisms are additionally involved in the observed effects. 相似文献
87.
本研究旨在探讨多巴胺D1受体在延髓离体脑片基本节律性呼吸放电调节中的作用。以改良的Kreb’s液(modified Kreb’s perfusion,MKS)恒温灌流Sprague—Dawley大鼠(0~3d)离体延髓脑片标本,稳定记录到与之相连的舌下神经根的呼吸节律性放电活动(respiratory rhythmical discharge activity,RRDA)后,第一组(n=5)先给予多巴胺D1受体特异性激动剂[cis-(±)-1-(Aminomethyl)-3,4-dihydro-3-phenyl-1H-2-Benzopyran-5,6-Diolhy,drochlo—ride,A68930]灌流10min,用MKS洗脱后,再给予多巴胺D1受体特异性拈抗剂[R(+)-7-Chloro-8-hydroxy-3-methyl—1—pheny1-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride,SCH-23390]灌流10min;第二组(n=5)先给予A68930持续灌流10min后再给予A68930+SCH-23390持续灌流10min;观察各时间点舌下神经根RRDA的变化。结果显示,给予A68930灌流后呼吸周期(respiratory cycle,RC)和呼气时程(expiratory time,TE)显著缩短,放电积分幅度(integral amplitude,IA)增加,吸气时程(inspiratory time,TI)无明显变化;给予SCH-23390后RC、TE显著延长、TI显著缩短、IA减小,而且A68930的作用可以被SCH.23390部分逆转。这些观察结果提示多巴胺D1受体参与了哺乳动物基本呼吸频率和幅度的调节。 相似文献
88.
89.
Ludwig Krabben Stefan Jehle Ekaterina N. Lyukmanova Alexander Arseniev Hartmut Oschkinat 《Journal of molecular biology》2009,390(4):662-19269
The contact area of neurotoxin II from Naja naja oxiana when interacting with the membrane-bound nicotinic acetylcholine receptor from Torpedo californica was determined by solid-state, magic-angle spinning NMR spectroscopy. For this purpose, the carbon signals for more than 90% of the residues of the bound neurotoxin were assigned. Differences between the solution and solid-state chemical shifts of the free and bound form of the toxin are confined to distinct surface regions. Loop II of the short toxin was identified as the main interaction site. In addition, loop III of neurotoxin II shows several strong responses defining an additional interaction site. A comparison with the structures of α-cobratoxin bound to the acetylcholine-binding protein from snail species Lymnaea stagnalis and Aplysia californica, and of α-bungarotoxin bound to an extracellular domain of an α-subunit of the receptor reveals different contact areas for long and short α-neurotoxins. 相似文献
90.
Sandra M. Dethlefsen Gerhard Raab Marsha A. Moses Rosalyn M. Adam Michael Klagsbrun Michael R. Freeman 《Journal of cellular biochemistry》1998,69(2):143-153
The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca2+ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143–153, 1998. © 1998 Wiley-Liss, Inc. 相似文献