scholarly journals Inhibition of inositol 1,4,5-trisphosphate 5-phosphatase by micromolar concentrations of disulfiram and its analogues

1993 ◽  
Vol 289 (3) ◽  
pp. 853-859 ◽  
Author(s):  
C J Fowler ◽  
G Brännström ◽  
P C Ahlgren ◽  
L Florvall ◽  
K E O Akerman
Keyword(s):  
Trypan Blue ◽  
Gh3 Cells ◽  
Trh Stimulation ◽  
Breakdown Rates ◽  
Inositol 1,4,5 Trisphosphate ◽  
Ic50 Values ◽  
Cellular Permeability ◽  
High Concentrations ◽  
Inositol Phospholipid Breakdown ◽  
Related Compounds

Following a preincubation period of 10 min, disulfiram and its analogues FLA 46, FLA 63, FLA 99, EWP 815 and EWP 840 inhibited the breakdown of 10 microM [3H]Ins(1,4,5)P3 by Ins(1,4,5)P3 5-phosphatase from GH3 cells, with IC50 values (in microM), for soluble/particulate enzymes respectively, of: disulfiram, 24/24; FLA 46, 23/30; FLA 63, 24/6; FLA 99, 50/48; EWP 815, 8/6; EWP 840, 11/8. The inhibition produced by FLA 99 was time-dependent in nature, although inhibition was found in the absence of a preincubation period. EWP 815 and EWP 840 were more potent inhibitors of Ins(1,4)P2 phosphatase than of Ins(1,4,5)P3 5-phosphatase. Thyrotropin-releasing hormone (TRH; 3/100 microM)-stimulated inositol phospholipid breakdown in prelabelled GH3 cells was inhibited by disulfiram (IC50 values 63/52 microM respectively), FLA 46 (89/110 microM), EWP 815 (83/71 microM) and EWP 840 (220/200 microM), without affecting basal breakdown rates. FLA 99 did not inhibit either basal or TRH-stimulated activity at any of the concentrations tested (30, 100 and 300 microM). [3H]Ins(1,4,5)P3 binding to its cerebellar receptor was not inhibited by any of the compounds over a concentration range of 3-300 microM, although an increased level of binding was seen at high concentrations. FLA 99 and EWP 840 increased the basal intracellular Ca2+ concentration in GH3 cells, but with no corresponding effect on the Ca2+ response to TRH stimulation. These compounds did not increase the cellular permeability to Trypan Blue, but did affect cell proliferation. It is concluded that disulfiram and related compounds produce dramatic effects on Ins(1,4,5)P3 metabolism in GH3 cells.

scholarly journals Separation of multiple isomers of inositol phosphates formed in GH3 cells

1987 ◽  
Vol 242 (2) ◽  
pp. 361-366 ◽  
Author(s):  
N M Dean ◽  
J D Moyer
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Inositol Phosphates ◽  
Thyrotropin Releasing Hormone ◽  
Gh3 Cells ◽  
Releasing Hormone ◽  
Inositol 1 ◽  
Trh Stimulation ◽  
Inositol 1,4,5 Trisphosphate

A high-performance-liquid-chromatography (h.p.l.c.) separation was developed, which resolves isomers of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) in a single run. In GH3 cells labelled with [3H]inositol, treated with Li+ and thyrotropin-releasing hormone (TRH), radiolabelled components identified as inositol 1-phosphate (I1P), inositol 2-phosphate (I2P), inositol 4-phosphate (I4P), inositol 1,4-bisphosphate [I(1,4)P2], inositol 1,3,4-trisphosphate [I(1,3,4)P3] and inositol 1,4,5-trisphosphate [I(1,4,5)P3] are present, as are multiple unidentified IP2 peaks. After TRH stimulation, both I1P and I4P increase, the increase in I4P preceding that of I1P; I(1,4)P2 and an unknown IP2 increase; and both I(1,3,4)P3 and I(1,4,5)P3 increase, the increase in I(1,4,5)P3 being rapid and transient, whereas the increase in I(1,3,4)P3 is slower and more sustained. The most rapidly appearing inositol phosphates produced after TRH stimulation are I(1,4)P2 and I(1,4,5)P3.

scholarly journals Preparation and characterization of a d-myo-inositol 1,4,5-trisphosphate-specific antibody

1995 ◽  
Vol 311 (3) ◽  
pp. 1009-1014 ◽  
Author(s):  
W R Shieh ◽  
C S Chen
Keyword(s):  
Binding Sites ◽  
Inositol Phosphates ◽  
Specific Binding ◽  
Affinity Purification ◽  
Competitive Elisa ◽  
Inositol 1,4,5 Trisphosphate ◽  
Ic50 Values ◽  
High Concentrations ◽  
Differential Affinity

Antibodies against Ins(1,4,5)P3 were raised by immunizing rabbits with two types of InsP3-BSA conjugates which were synthesized by covalently coupling Ins(1,4,5)P3 to the carrier protein via alkyl linkages. The anti-Ins(1,4,5)P3 antibody was detected by a novel ELISA using Ins(1,4,5)P3-immobilized microtitre plates. Both antiserum preparations showed specific binding with Ins(1,4,5)P3, with titres of 1:4000. Most inositol phosphates, including Ins1P, Ins(4,5)P2, Ins(1,3,4)P3, Ins(1,5,6)P3, Ins(1,2,5,6)P1, Ins(3,4,5,6)P4, Ins(1,3,4,5,6)P5, InsP6, and PtdIns(4,5)P2, did not exhibit significant molecular interactions with the antibodies. Ins(1,3,4,5)P4, however, cross-reacted with these antibodies with one-third of the affinity as that of Ins(1,4,5)P3, in part due to the largely shared structural motifs. The differential affinity was significantly improved by affinity purification on Ins(1,4,5)P3-agarose. The affinity-purified antibody displayed IC50 values of 12 nM and 730 nM for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 respectively, according to a competitive ELISA; these values are in line with those reported for the Ins(1,4,5)P3 receptor. The modes of ligand recognition at the binding sites of these two types of biomolecules are, however, different. Moreover, although the ligand binding was interfered with by multivalent anions such as ATP4-, HPO4(3-) and SO4(2-) at high concentrations, no inhibition was noted with heparin, an antagonist of the Ins(1,4,5)P3 receptor.

FURTHER STUDIES ON THE DUAL REQUIREMENT FOR PHENYLALANINE AND TYROSINE IN TISSUE CULTURE

1961 ◽  
Vol 39 (5) ◽  
pp. 925-932 ◽  
Author(s):  
Helen J. Morton ◽  
Joseph F. Morgan
Keyword(s):  
Amino Acids ◽  
Tissue Culture ◽  
Chick Embryo ◽  
Heart Cells ◽  
Present System ◽  
Chick Heart ◽  
High Concentrations ◽  
Chick Embryo Heart ◽  
Embryo Heart ◽  
Related Compounds

Seventeen structurally related compounds were tested for their ability to substitute for phenylalanine or tyrosine in the nutrition of chick embryo heart fragments. DL-Alanyl-DL-phenylalanine replaced phenylalanine. All other compounds had negligible effects, and most were toxic at high concentrations. β-Phenylserine, a phenylalanine antagonist, actually prolonged the survival of chick heart cells but only if both phenylalanine and tyrosine were present. Similarly, optimal reversal of β-phenylserine toxicity was dependent on the presence of both amino acids. Although phenylalanine and tyrosine are not interconvertible in the present system, it has been shown that three phenylalanine antagonists, p-fluorophenylalanine, β-2-thienylalanine, and β-phenylserine, can be identified by their relationship to tyrosine, rather than to phenylalanine.

α-Glucosidase Inhibitory and Anti-Inflammatory Coumestans from the Roots of Dolichos trilobus

Planta Medica ◽  
2018 ◽  
Vol 85 (02) ◽  
pp. 112-117 ◽  
Author(s):  
Meng-Yuan Jiang ◽  
Ming Luo ◽  
Kai Tian ◽  
Yan-Hong Li ◽  
Jing-Xian Sun ◽  
...  
Keyword(s):  
Spectroscopic Data ◽  
Data Interpretation ◽  
Raw 264.7 Cells ◽  
Raw 264.7 ◽  
Nitric Oxide Production ◽  
Murine Macrophage ◽  
Cytotoxic Effects ◽  
Ic50 Values ◽  
Anti Inflammatory ◽  
Related Compounds

AbstractFour new coumestans dolichosins A – D (1–4) were isolated from the roots of Dolichos trilobus, together with four known compounds: isosojagol (5), phaseol (6), psoralidin (7), and 4″,5″-dehydroisopsoralidin (8). Their structures were elucidated on the basis of spectroscopic data interpretation, mass spectrometric analyses, and the comparison with literature data of related compounds. The anti-inflammatory activity of these compounds (1–8) was evaluated through the inhibition of nitric oxide production in lipopolysaccharide-activated murine macrophage RAW 264.7 cells, in which compounds 1 and 6 displayed moderate inhibitory activity and no cytotoxic effects. In a α-glucosidase inhibitory assay, compounds 1 and 5–8 exhibited appreciable inhibition on α-glucosidase. Especially compounds 1, 7, and 8 showed IC50 values lower than 20.0 µM.

scholarly journals Analogs of the Autoinducer 3-Oxooctanoyl-Homoserine Lactone Strongly Inhibit Activity of the TraR Protein ofAgrobacterium tumefaciens

1998 ◽  
Vol 180 (20) ◽  
pp. 5398-5405 ◽  
Author(s):  
Jun Zhu ◽  
John W. Beaber ◽  
Margret I. Moré ◽  
Clay Fuqua ◽  
Anatol Eberhard ◽  
...  
Keyword(s):  
Homoserine Lactone ◽  
Mass Action ◽  
Wild Type ◽  
Dna Binding Sites ◽  
Agonistic Activity ◽  
High Concentrations ◽  
Mediate Cell ◽  
Potent Antagonist ◽  
Almost All ◽  
Related Compounds

ABSTRACT The TraR and TraI proteins of Agrobacterium tumefaciensmediate cell-density-dependent expression of the Ti plasmidtra regulon. TraI synthesizes the autoinducer pheromoneN-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL), while TraR is an 3-oxo-C8-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C8-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only 3-oxo-C8-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive. Furthermore, many of these compounds were potent antagonists. In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist. We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action. Wild-type A. tumefaciens released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. However, under all conditions tested, 3-oxo-C8-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with tra gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C8-HSL significantly stimulates tra gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers. When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.

scholarly journals Slow kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release: is the release ‘quantal’ or ‘non-quantal’?

1997 ◽  
Vol 323 (1) ◽  
pp. 123-130 ◽  
Author(s):  
Ludwig MISSIAEN ◽  
Humbert DE SMEDT ◽  
Jan B. PARYS ◽  
Ilse SIENAERT ◽  
Henk SIPMA ◽  
...  
Keyword(s):  
Low Dose ◽  
Slow Phase ◽  
Quantal Response ◽  
Published Evidence ◽  
A7r5 Cells ◽  
Slow Kinetics ◽  
Inositol 1,4,5 Trisphosphate ◽  
Long Time ◽  
High Concentrations ◽  
Kinetics Of

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores is generally assumed to be a ‘quantal’ process because low InsP3 concentrations mobilize less Ca2+ than high concentrations and a submaximal concentration does not release all the InsP3-mobilizable Ca2+. However, some recent reports questioned the generally accepted view that a low dose of InsP3 is unable to empty the whole store. We have now challenged the stores of permeabilized A7r5 cells in InsP3 for much longer periods than previously reported, to assess directly whether the slow phase of the release would empty the whole store (a non-quantal response) or only a fraction of it (a quantal response). Addition of a maximal [InsP3] at the end of a prolonged (92 min) stimulation with a submaximal [InsP3] resulted in additional Ca2+ release. Experiments in which the stores were challenged with different submaximal InsP3 concentrations for long time periods revealed that a lower [InsP3] never released the same amount of Ca2+ as a higher [InsP3]. This quantal pattern of Ca2+ release occurred both at 25 °C and at 4 °C. There was a time-dependent increase in the fraction of Ca2+ recruited by the lower compared with the higher [InsP3]. This recruitment of Ca2+ persisted if the [InsP3] was decreased, but was largely prevented by palmitoyl-CoA, a potent blocker of the luminal Ca2+ translocation between individual store units. ATP, in the presence of InsP3, released Ca2+ under conditions permitting the recruitment of no additional InsP3 receptors, indicating that an all-or-none emptying of a fraction of the stores cannot be the only mechanism responsible for quantal Ca2+ release in A7r5 cells. We conclude that some of the previously published evidence for a non-quantal Ca2+ release pattern should be reinterpreted.

Effects of IAA in combination with FSH on in vitro culture of ovine preantral follicles

Zygote ◽  
2009 ◽  
Vol 18 (1) ◽  
pp. 89-92 ◽  
Author(s):  
Sonia H.F. Costa ◽  
Regiane R. Santos ◽  
Davide Rondina ◽  
Evelyn R. Andrade ◽  
Otávio M. Ohashi ◽  
...  
Keyword(s):  
Trypan Blue ◽  
Follicle Stimulating Hormone ◽  
Follicular Development ◽  
Free Medium ◽  
Minimum Essential Medium ◽  
Viability Test ◽  
Preantral Follicles ◽  
High Concentrations ◽  
Indole 3 Acetic Acid

SummaryOvarian cortical fragments from five adult ewes were in vitro cultured for 1, 3 or 5 days in the presence of minimum essential medium either supplemented or not by follicle-stimulating hormone (FSH) (100 ng/ml) or indole-3-acetic acid (IAA) (10, 20, 40 or 100 ng/ml), alone or in combination. After in vitro culture, ovarian fragments were submitted to follicular isolation and viability test was performed using trypan blue. Addition of IAA (10 ng/ml) to a free-FSH medium resulted in the highest percentages of viable follicles, but was progressively deleterious in higher concentrations (20, 40 and 100 ng/ml) if in absence of FSH. Follicular development was observed only when FSH was added to an IAA-free medium. In conclusion, IAA at a concentration of 10 ng/ml increases follicular survival in vitro. However, at high concentrations (20, 40 or 100 ng/ml), this auxin may be deleterious to preantral follicles, the addition of FSH to the medium being necessary.

scholarly journals Electroporation can cause artefacts due to solubilization of cations from the electrode plates. Aluminum ions enhance conversion of inositol 1,3,4,5-tetrakisphosphate into inositol 1,4,5-trisphosphate in electroporated L1210 cells

1991 ◽  
Vol 277 (3) ◽  
pp. 883-885 ◽  
Author(s):  
J W Loomis-Husselbee ◽  
P J Cullen ◽  
R F Irvine ◽  
A P Dawson
Keyword(s):  
Experimental System ◽  
Experimental Medium ◽  
Assay Medium ◽  
L1210 Cells ◽  
Aluminum Ions ◽  
Inositol 1,4,5 Trisphosphate ◽  
The Difference ◽  
High Concentrations ◽  
As If ◽  
In Cells

1. In electroporated L1210 cells, Ins(1,3,4,5)P4 causes Ca2+ release, owing to its conversion into Ins(1,4,5)P3, but this does not happen in cells permeabilized by digitonin treatment [Cullen, Irvine, Drøbak & Dawson (1989) Biochem. J. 259, 931-933]. 2. If the assay medium is subjected to electroporation by using a commercially available electroporation apparatus and then the cells are added and permeabilized with digitonin, the cells behave as if they had been electroporated. 3. Electroporation causes the release of high concentrations of Al3+ into the experimental medium, and addition of these concentrations of Al3+ into the experimental medium mimics the effect of electroporation on the conversion of Ins(1,3,4,5)P4 into Ins(1,4,5)P3. 4. It is concluded that the difference between electroporated and digitonin-permeabilized L1210 cells in this experimental system can be attributed to dissolution of Al3+ from the electroporation cuvette. Al3+ contamination may thus be a serious problem when using this apparatus.

scholarly journals Kite-Shaped Molecules Block SARS-CoV-2 Cell Entry at a Post-Attachment Step

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2306
Author(s):  
Shiu-Wan Chan ◽  
Talha Shafi ◽  
Robert C. Ford
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Small Molecules ◽  
Cell Entry ◽  
Human Cell Lines ◽  
Structural Homology ◽  
Virus Attachment ◽  
Ic50 Values ◽  
Coronavirus Infection ◽  
Related Compounds ◽  
Infectious Cycle

Anti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50 values in the 2–5 μM range, were identified. Further studies demonstrated that these “kite-shaped” molecules were surprisingly specific for SARS-CoV-1 and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover, the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.

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