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A detailed NMR study of the thiopeptide amythiamicin D establishes its solution conformation and the presence of a single intramolecular hydrogen bond involving NH13 and O28, and also provides the first evidence for self-association of thiopeptides in solution.
The effect of encapsulation by cucurbiturils Q[7] and Q[8] on the rate of reaction of the anti-cancer dinuclear platinum complex trans-[{PtCl(NH3)2}2(micro-NH2(CH2)8NH2)]2+ with the model biological nucleophiles glutathione and cysteine has been examined by NMR spectroscopy. It was expected that the octamethylene linking chain would fold inside the cucurbituril host and hence position the reactive platinum centres close to the cucurbituril portals, and thereby, confer resistance to degradation by biological nucleophiles. The upfield shifts of the resonances from the methylene protons in the linking ligand observed in 1H NMR spectra of the platinum complex upon addition of either Q[7] or Q[8] indicate that the cucurbituril is positioned over the linking ligand, with the Pt(II) centres projecting out of the portal. Furthermore, the relative changes in chemical shift of the methylene resonances suggest that the octamethylene linking chain folds within the cucurbituril cavity, particularly in Q[8]. Simple molecular models, based on the observed relative changes in chemical shift, could be constructed that were consistent with the proposed folding of the linking ligand within the cucurbituril cavity. Encapsulation by Q[7] was found to reduce the rate of reaction of the platinum complex with glutathione. Encapsulation by Q[7] and Q[8] was also found to reduce the rate of reaction of the platinum complex with cysteine, with Q[8] slowing the reaction to a greater extent than Q[7], consistent with the inferred encapsulation geometries. Encapsulation of dinuclear platinum complexes within the cucurbituril cavity may provide a novel way of reducing the reactivity and degradation of these promising chemotherapeutic agents with blood plasma proteins.
The reaction of a heteroligated Rh(I) bimetallic macrocycle with rigid ditopic ligands (1,4-dicyanobenzene, 4-4'-dicyanobiphenyl, or dipyridyl terminated salen ligand 5) results in the formation of tetrametallic rectangular box complexes.
A new polyammonium receptor is able to selectively recognise and sense ATP among triphosphate nucleotides, thanks to ATP-induced quantitative quenching of its fluorescence emission.
A protocol for the construction of poly-oxazoles with consecutive 2,4'-linkages is described, and has afforded an efficient route to a penta-oxazole which demarcates a route to telomestatin and related macrocyclic poly-oxazole systems.
The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.
Physical coupling between inositol 1,4,5-trisphosphate (IP(3)) receptors and transient receptor potential (Trp) channels has been demonstrated in both transfected and normal cells as a candidate mechanism for the activation of store-mediated Ca(2+) entry (SMCE). We have investigated the properties of the coupling between the type II IP(3) receptor and naturally expressed human Trp1 (hTrp1) in human platelets. Treatment with xestospongin C, an inhibitor of IP(3) receptor function, abolished SMCE and coupling between the IP(3) receptor and hTrp1. The coupling was activated by depletion of the intracellular Ca(2+) stores, and was reversed by refilling of the stores. We have also examined the role of actin filaments in the activation and maintenance of the coupling. Stabilization of the cortical actin network with jasplakinolide prevented the coupling, indicating that, as with secretion, the actin filaments at the cell periphery act as a negative clamp which prevents constitutive coupling. In addition, the actin cytoskeleton plays a positive role, since disruption of the actin network inhibited the coupling when the Ca(2+) stores were depleted. These results provide strong evidence for the activation of SMCE by a secretion-like coupling mechanism involving a reversible association between IP(3) receptors and hTrp1 in normal human cells.
Glucagon-like peptide-1 (GLP-1) is a potent regulator of glucose-stimulated insulin secretion whose mechanisms of action are only partly understood. In the present paper, we show that at low (3 mM) glucose concentrations, GLP-1 increases the free intramitochondrial concentrations of both Ca(2+) ([Ca(2+)](m)), and ATP ([ATP](m)) in clonal MIN6 beta-cells. Suggesting that cAMP-mediated release of Ca(2+) from intracellular stores is responsible for these effects, increases in [ATP](m) that were induced by GLP-1 were completely blocked by the Rp isomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), or by chelation of intracellular Ca(2+). Furthermore, inhibition of Ins(1,4,5) P (3) (IP(3)) receptors with xestospongin C, or application of ryanodine, partially inhibited GLP-1-induced [ATP](m) increases, and the simultaneous blockade of both IP(3) and ryanodine receptors (RyR) completely eliminated the rise in [ATP](m). GLP-1 appeared to prompt Ca(2+)-induced Ca(2+) release through IP(3) receptors via a protein kinase A (PKA)-mediated phosphorylation event, since ryanodine-insensitive [ATP](m) increases were abrogated with the PKA inhibitor, H89. In contrast, the effects of GLP-1 on RyR-mediated [ATP](m) increases were apparently mediated by the cAMP-regulated guanine nucleotide exchange factor cAMP-GEFII, since xestospongin C-insensitive [ATP](m) increases were blocked by a dominant-negative form of cAMP-GEFII (G114E,G422D). Taken together, these results demonstrate that GLP-1 potentiates glucose-stimulated insulin release in part via the mobilization of intracellular Ca(2+), and the stimulation of mitochondrial ATP synthesis.
Differential utilization of intracellular Ca2+ stores with specific functional characteristics could be a potential mechanism for coupling various stimuli to specific cellular responses. In the goldfish pituitary, both gonadotropes and somatotropes possess multiple intracellular Ca2+ stores that are differentially coupled to agonist-evoked exocytosis. We investigated the role of ryanodine receptor/Ca2+-release channels (RyR) in basal and gonadotropin-releasing hormone (GnRH)-evoked hormone secretion from cultured gonadotropes and somatotropes using radioimmunoassay for gonadotropin (GTH-II) and growth hormone (GH). As is the case in vivo, the basal and evoked secretion of both hormones varied with seasonal reproductive status. GnRH-stimulated hormone release was three-fold higher in cells from sexually mature animals compared to those in a sexually regressed state. Nanomolar doses of ryanodine evoked significant GTH-II and GH secretion, suggesting that ryanodine-sensitive Ca2+ stores can couple to exocytosis in both cell types. In gonadotropes, 10 microM ryanodine abolished cGnRH-II-evoked GTH-II release in both sexually mature and sexually regressed fish, while sGnRH signalling was mediated by ryanodine-sensitive Ca2+ stores in cells from sexually regressed fish only. Ryanodine-sensitive Ca2+ stores in somatotropes were only involved in cGnRH-II-stimulated GH release during gonadal regression. In contrast, sGnRH-stimulated, but not cGnRH-II-stimulated, GH release was significantly reduced by 1 microM xestospongin C. Although hormone release stimulated by mobilizing caffeine-sensitive Ca2+ pools was also markedly seasonal, it was largely independent of ryanodine-sensitive Ca2+ stores. Ryanodine-sensitive Ca2+ stores in both cell types are not active downstream of ionomycin, BayK 8644, protein kinase C or cyclic adenosine monophosphate signalling pathways, suggesting difference from a classical Ca2+-induced Ca2+ release system. Ours study is the first to suggest that RyR2 may be involved in the seasonal plasticity of pituitary function, which may be related to cyclic changes observed in reproduction and growth.
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Xestospongins, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge, Xestospongia species, are potent blockers of the inositol 1,4,5-trisphosphate (IP(3))-induced Ca2+ release in bi-directional Ca2+-flux conditions. We have now studied the effects of xestospongin C on the (45)Ca2+ uptake and the uni-directional (45)Ca2+ efflux in permeabilized A7r5 smooth-muscle cells. Xestospongin C not only inhibits the IP(3)-induced Ca2+ release, but is also an equally potent blocker of the endoplasmic-reticulum Ca2+ pump, while it has no effect on the passive Ca2+ leak. The inhibition of the IP(3) receptor did not depend on the IP(3), Ca2+ or ATP concentration. Xestospongin C can, therefore, not be considered as a selective blocker of IP(3) receptors.
The recent establishment of a robust hepatitis C virus (HCV) cell culture system permits analysis of virus-host interactions during HCV infection. Here, we report that HCV genotype 2a (JFH-1) infection fails to induce IFN-beta or IFN-stimulated gene expression in Huh-7 cells, and that it blocks IFN-beta and IFN-stimulated gene production after transfection of synthetic dsRNA. Overexpression of individual components of the dsRNA-signaling pathway in HCV-infected and uninfected cells indicates that HCV inhibits IFN-beta promoter activity by inactivating the mitochondrial antiviral signaling protein/IFN-beta promoter stimulator 1 (MAVS/IPS-1), while leaving the IFN-induced Janus kinases-signal transducers and activators of transcription (JAK-STAT) signaling pathway intact. We also show that MAVS/IPS-1-dependent IFN-beta promoter activity in HCV-infected cells is fully restored by the nonstructural protein 3 (NS3) protease inhibitor BILN2061. In contrast, synthetic dsRNA-induced IFN-beta promoter activity is not restored by BILN2061, although it is partially restored by overexpression of RIG-I. These results support recently reported evidence that the HCV NS3 protease blunts the ability of HCV to induce IFN-beta promoter activity by proteolytically cleaving MAVS/IPS-1. The results also suggest that HCV blocks the synthetic dsRNA-induced signaling pathway at a point upstream of MAVS/IPS-1, and that it does so by an NS3-independent mechanism.
Recent studies suggest a role for autocrine insulin signaling in beta cells, but the mechanism and function of insulin-stimulated Ca(2+) signals is uncharacterized. We examined Ca(2+)-dependent insulin signaling in human beta cells. Two hundred nanomolar insulin elevated [Ca(2+)](c) to 284 +/- 27 nM above baseline in approximately 30% of Fura-4F-loaded cells. Insulin evoked multiple Ca(2+) signal waveforms, 60% of which included oscillations. Although the amplitude of Ca(2+) signals was dose-dependent between 0.002 and 2,000 nM, the percentage of cells responding was highest at 0.2 nM insulin, suggesting the interaction of stimulatory and inhibitory pathways. Ca(2+)-free solutions did not affect the initiation of insulin-stimulated Ca(2+) signals, but abolished the second phase of plateaus/oscillations. Likewise, inositol 1,4,5-trisphosphate (IP(3)) receptor antagonists xestospongin C and caffeine selectively blocked the second phase, but not the initiation of insulin signaling. Thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ) blocked insulin signaling, implicating sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA)-containing Ca(2+) stores. Insulin-stimulated Ca(2+) signals were insensitive to ryanodine. Injection of the CD38-derived Ca(2+) mobilizing metabolite, nicotinic acid-adenine dinucleotide phosphate (NAADP), at nanomolar concentrations, evoked oscillatory Ca(2+) signals that could be initiated in the presence of ryanodine, xestospongin C, and Ca(2+)-free solutions. Desensitizing concentrations of NAADP abolished insulin-stimulated Ca(2+) signals. Insulin-stimulated Ca(2+) signals led to a Ca(2+)-dependent increase in cellular insulin contents, but not secretion. These data reveal the complexity of insulin signal transduction and function in human beta cells and demonstrate functional NAADP-sensitive Ca(2+) stores in a human primary cultured cell type.
In nonexcitable cells, stimulation by high agonist concentrations typically produces a biphasic increase in cytosolic Ca(2+) ([Ca(2+)](i)). This response is characterized by a transient initial increase because of intracellular Ca(2+) release followed by a sustained elevation which varies in amplitude depending on the nature of the stimulus. In contrast, low-level stimulation often evokes oscillatory changes in [Ca(2+)](i). The specific information provided by repetitive [Ca(2+)](i) spikes appears to be encoded in the frequency rather than in the amplitude of [Ca(2+)](i) oscillations. The specific, membrane-permeable inositol 1,4, 5-trisphosphate (Ins-1,4,5-P(3)) receptor blocker Xestospongin C (XeC, 2-20 microM) was used to affect [Ca(2+)](i) signaling in human aortic endothelial cells (HAEC) during an established response to low-level (1 microM) histamine stimulation. XeC produced a dose-dependent decrease in the frequency of [Ca(2+)](i) oscillations during histamine stimulation without affecting oscillation amplitude. Histamine stimulated a 14-fold increase in NF-kappaB-chloramphenicol acetyltransferase reporter gene activity that was dose-dependently decreased by XeC. Thus, during low-level agonist stimulation, [Ca(2+)](i) oscillation frequency regulates nuclear transcription in HAEC.
Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J. A. , Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca(2+)](i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.
Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.
Reperfusion of ischemic myocardium leads to a local burst of free radicals, increased [Ca(2+)](i), and the release of proinflammatory cytokines. The purpose of this study was to determine whether brief exposure of cardiac fibroblasts to H(2)O(2) is associated with transient changes in [Ca(2+)](i) levels and whether this stimulus is sufficient to induce interleukin-6 (IL-6) expression. Cardiac derived fibroblasts were isolated from adult male rats and cultured under standard conditions. Individual coverslip-attached fibroblasts were loaded with the calcium probe Fura-2/AM and exposed to a single 3-min pulse of 100 microm H(2)O(2). In addition, low passage cultures were exposed to a pulse of H(2)O(2) and assayed for IL-6 expression. A brief exposure of H(2)O(2) led to a large intracellular Ca(2+) transient with a mean transient magnitude of 318 +/- 28 nm (mean +/- S.D., n = 12). Stimulation in the absence of [Ca(2+)](o) led to a 59% reduction in mean transient magnitude (129 +/- 23 nm, n = 10, p < 0.001), whereas pretreatment with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C resulted in a 37% reduction (199 +/- 25 nm, n = 10, p < 0.01). Cells treated with xestospongin C and stimulated in the absence of [Ca(2+)](o) did not exhibit a Ca(2+) transient. Time-dependent IL-6 release was significantly elevated by 4 h (368 +/- 64 pg/mg protein, p < 0.01) and increased further by 24 h (1030 +/- 76 pg/mg protein). The depletion of cellular Ca(2+) by pretreatment with thapsigargin in the absence of [Ca(2+)](o) attenuated H(2)O(2)-induced IL-6 mRNA expression while blocking protein release. These data show that the exposure of cardiac fibroblasts to a brief pulse of physiological levels of H(2)O(2) resulted in a large Ca(2+) transient with intracellular and extracellular Ca(2+) contributions. Furthermore, brief H(2)O(2) exposure led to calcium-dependent IL-6 expression.