Calculated IC50 Values Research Articles

Molecular Recognition of the Protein Phosphatase 1 Glycogen Targeting Subunit by Glycogen Phosphorylase

Disrupting the interaction between glycogen phosphorylase and the glycogen targeting subunit (G(L)) of protein phosphatase 1 is emerging as a novel target for the treatment of type 2 diabetes. To elucidate the molecular basis of binding, we have determined the crystal structure of liver phosphorylase bound to a G(L)-derived peptide. The structure reveals the C terminus of G(L) binding in a hydrophobically collapsed conformation to the allosteric regulator-binding site at the phosphorylase dimer interface. G(L) mimics interactions that are otherwise employed by the activator AMP. Functional studies show that G(L) binds tighter than AMP and confirm that the C-terminal Tyr-Tyr motif is the major determinant for G(L) binding potency. Our study validates the G(L)-phosphorylase interface as a novel target for small molecule interaction.

In vitro oxime protection of human red blood cell acetylcholinesterase inhibited by diisopropyl‐fluorophosphate

Oximes are enzyme reactivators used in treating poisoning with organophosphorus cholinesterase (AChE) inhibitors. The oxime dose which can be safely administered is limited by the intrinsic toxicity of the substances such as their own AChE-inhibiting tendency. Clinical experience with the available oximes is disappointing. To meet this need, new AChE reactivators of potential clinical utility have been developed. The purpose of the study was to estimate in vitro both the intrinsic toxicity and the extent of possible protection conferred by established (pralidoxime, obidoxime, HI-6, methoxime, trimedoxime) and experimental (K-type) oximes, using diisopropyl-fluoro-phosphate (DFP) as an AChE inhibitor. The IC50 of DFP against human red blood cell AChE was determined ( approximately 120 nm). Measurements were then repeated in the presence of increasing oxime concentrations, leading to an apparent increase in DFP IC50. Calculated IC50 values were plotted against oxime concentrations to obtain an IC50 shift curve. The slope of this shift curve (tan alpha) was used to quantify the magnitude of the protective effect (nm IC50 increase per microm oxime). We show that, in the case of a linear relationship between oxime concentration and IC50, the binding constant K, determined using the Schild equation, equals IC50/DFP/tan alpha. Based on the values of tan alpha and of the binding constant K, some of the new K-oxime reactivators are far superior to pralidoxime (tan alpha = 0.8), obidoxime (1.5), HI-6 (0.8), trimedoxime (2.9) and methoxime (5.9), with K-107 (17), K-108 (20), and K-113 (16) being the outstanding compounds.

Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.

Inhibition of Reactive Nitrogen Species in Vitro and ex Vivo by Trypsin Inhibitor from Sweet Potato ‘Tainong 57' Storage Roots

Peroxynitrite (ONOO-), formed from a reaction of superoxide and nitric oxide, is one of the most potent cytotoxic species known to oxidize cellular constituents including essential proteins, lipids, and DNA. ONOO- induces cellular and tissue injury, resulting in several human diseases such as Alzheimer's disease, atherosclerosis, and stroke. Due to the lack of endogenous enzymes responsible for ONOO- scavenging activity, finding a specific ONOO- scavenger is of considerable importance. In this study, the ability of trypsin inhibitor (TI), isolated from sweet potato storage roots (SPTI), to scavenge *ON and ONOO- was investigated. The data obtained show that TI generated a dose-dependent inhibition on production of nitrite and superoxide radicals. The IC50 value of TI on superoxide radical was 143.2 +/- 4.29 microg/mL. SOD activity staining was used to confirm SOD activity of SPTI. SPTI also caused a dose-dependent inhibition of the oxidation of dihydrorhodamine 123 (DHR) by peroxynitrite. A calculated IC50 value of 809.1 +/- 32.36 microg/mL was obtained on the inhibition of peroxynitrite radical. Spectrophotometric analyses revealed that TI suppressed the formation of ONOO--mediated tyrosine nitration through an electron donation mechanism. In further studies, TI also showed a significant ability to inhibit nitration of bovine serum albumin (BSA) in a dose-dependent manner. In vivo TI inhibited lipopolysaccharide-induced nitrite production in macrophages in a concentration-dependent manner with an IC50 value of 932.8 +/- 29.85 microg/mL. The present study suggested that TI had an efficient reactive nitrogen species scavenging ability. TI might be a potential effective NO and ONOO- scavenger useful for the prevention of NO- and ONOO--involved diseases.

Inhibition of Bone Morphogenetic Protein 1 by Native and Altered Forms of α2-Macroglobulin

The four mammalian bone morphogenetic protein 1 (BMP1)-like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein alpha(2)-macroglobulin is unable to inhibit the astacin-like proteinases meprin alpha and meprin beta, we herein demonstrate alpha(2)-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the alpha(2)-macroglobulin "bait" region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I-III. alpha(2)-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified alpha(2)-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced approximately 24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of alpha(2)-macroglobulin in the treatment of fibrotic conditions.

Synthesis and Characterization of Oligodeoxynucleotides Containing Naphthyridine:Imidazopyridopyrimidine Base Pairs at their Sticky Ends. Application as Thermally Stabilized Decoy Molecules

We describe the synthesis and properties of oligodeoxynucleotides (ODNs) containing 1,8-naphthyridine C-nucleoside (Na-NO) and imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleoside (Im-ON) at the termini. The modified ODNs were more resistant (6 to 40 times) than natural DNA to snake venom phosphodiesterase (SVPD). Although incorporation of one pair each of Na-NO:Im-ON on the sticky ends of the duplex was insufficient for thermal stabilization (+2.5 degrees C per pair relative to the G:C pair), the duplex containing two consecutive Na-NO:Im-ON pairs at its sticky ends was markedly stabilized thermally. The stabilizing effect of the incorporation of additional Na-NO:Im-ON pairs is estimated to be +7.8 degrees C per pair. Application as thermally stabilized decoy molecules to NF-kappaB (p50) was also demonstrated. The DNA duplexes containing the Na-NO:Im-ON pairs (ODN I:ODN II and ODN III:ODN IV) acted as competitors to the natural NF-kappaB-binding duplex (ODN V: ODN VI), and the calculated IC50 values of ODN I:ODN II and ODN III:ODN IV were 20.1+/-13.3 and 10.9+/-4.8 nM, respectively, greater than that of ODN V:ODN VI.

Antitumor and Immunostimulatory Activity of Two Chromones and Other Constituents from Cassia Petersiana

Phytochemical and biological investigation of the leaves of Cassia petersiana afforded two new chromones (1, 2), in addition, to the known glyceryl-1-hexacosanoate (3) and stigmasterol-3-O-β-D-glucoside (4). The structures of the compounds were determined by comprehensive NMR studies, including DEPT, COSY, HMQC, HMBC and IR spectroscopy and MS. 1–4 were investigated against different types of cell lines, including solid tumor cells (Hep-G2, and MCF-7 cells) and leukemia (1301) cells for their cytotoxic effects. 1–3 possessed a dose-dependent cytotoxic effect against Hep-G2 cells, but in relatively high concentrations; 4 was the most cytotoxic with the lowest IC50 value of 82.7 μM. The calculated IC50 values against MCF-7 cells were 112.2 μM, 143.7 μM, 68.1 μM, and 114.3 μM for 1, 2, 3, and 4, respectively. The alteration in the macrophage proliferation index, using Raw 264.7 cells, was monitored. 1 and 3 were the highest stimulators of macrophage proliferation in a dose-dependent manner, whereas 2 and 4 showed a peak point of stimulation at 20 μM. The effect of these compounds on pre-induced NO was explored. 1–4 inhibited the LPS-induced NO, with inhibition percentages of 80.5%, 89.3%, 82.1%, and 92.1%, respectively, at a concentration of 20 μM. The antioxidant capacity of 1–4, using the DPPH assay was also investigated. 1–3 possessed weak scavenging activity; while 4 had an effective SC50 value as low as 36 μM. These results indicated that 4 possessed the highest anti-tumor, immunoproliferative, macrophage proliferation, anti-inflammatory, and antioxidant activities.

Selective Excision of Chain-terminating Nucleotides by HIV-1 Reverse Transcriptase with Phosphonoformate as Substrate

A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on PP(i)- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP(i)- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT.template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP(i) or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.

Five oximes (K-27, K-33, K-48, BI-6 and methoxime) in comparison with pralidoxime:in vitro reactivation of red blood cell acetylcholinesterase inhibitied by paraoxon

Oximes are cholinesterase reactivators of use in poisoning with organophosphorus compounds. Pralidoxime (PRX) is used clinically as an adjunct to atropine in such exposure. Clinical experience with PRX (and other oximes) is, however, disappointing and routine use has been questioned. In addition it is known that oximes are not equally effective against all existing organophosphorus compounds. There is a clear demand for 'broad spectrum' cholinesterase reactivators with a higher efficacy than PRX. Over the years new reactivators of cholinesterase of potential clinical utility have been developed. Their chemical structures were derived from those of existing esterase reactivators, especially pralidoxime, obidoxime and HI-6. The purpose of the study was to quantify in vitro the extent of oxime (pralidoxime, K-27, K-33, K-48, methoxime and BI-6) conferred protection, using paraoxon as an inhibitor. Paraoxon (POX), the active metabolite of parathion (O,O-diethyl-O-p-nitro-phenyl phosphorothioate) is a non-neuropathic organophosphate. Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood were measured photometrically in the presence of different POX concentrations and the IC50 was calculated. Determinations were repeated in the presence of increasing oxime concentrations. The IC50 of POX increases with the oxime concentration in a linear manner. The calculated IC50 values were plotted against the oxime concentrations to obtain an IC50 shift curve. The slope of the shift curve (tg alpha) was used to quantify the magnitude of the protective effect (nm IC50 increase per microm reactivator). Based on our determinations the new K series of reactivators is far superior to pralidoxime, methoxime and BI-6, K-27 being the outstanding compound with a tg alpha value of 3.7 (nm IC50 increase per microm reactivator) which is approximately 13 times the reactivator ability of PRX. In general there is an (expected) inverse relationship between the binding constant K and the slope of the IC50 shift curve (tg alpha) for all oximes examined. K-27 (the most protective substance judging by the tg alpha) has the lowest K value (highest affinity). In vivo testing of the new oximes as an organophosphate protective agent is necessary.

Molecular mechanisms of inhibitory activities of tanshinones on Lipopolysaccharide-lnduced nitric oxide generation in RAW 264.7 cells

The effects of four tanshinones isolated from Tanshen (the root of Salvia miltiorrhiza Bunge, Labiatae) were tested for their inhibition of nitric oxide production in macrophage cells, and the underlying molecular mechanisms studied. Of the four tanshinones used, 15, 16-dihydrotanshinone-I, tanshinone-IIA and cryptotanshinone, but not tanshinone I, demonstrated significant inhibition of the LPS-induced nitric oxide production in RAW 264.7 cells, with calculated IC50 values of 5, 8, and 1.5 microM, respectively. Tanshinones exerted inhibitory activities on the LPS-induced nitric oxide production only when applied concurrently with LPS, and tanshinone-IIA and cryptotanshinone were found to inhibit LPS-induced NF-kappaB mobilization and extracellular-regulated kinase (ERK) activation, respectively. These results suggest that tanshinones inhibit LPS-induced nitric oxide generation by interfering with the initial stage of LPS-induced expression of certain genes. NF-kappaB and ERK could be the molecular targets for tanshinones for the inhibition of LPS-induced nitric oxide production in macrophage cells.

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a beta-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic alpha-methylene-gamma-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a beta-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 microg/ml: (1) 93.4+/-6.62%, (2) 98,5+/-1.19%, 5 14.7+/-2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose-response relationship in the range of 3.33-100 microg/ml. According to the calculated IC50 values the highest inhibitory activity was observed for the depsidone 1 (IC50 = 28.5 microM) followed by 2 (IC50 = 77.0 microM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC50 = 24.6 microM).

Pharmacodynamic enhancement of the anti-platelet antibody Fab abciximab by site-specific pegylation

Monoclonal antibodies have been firmly established as human therapeutics. Their high affinity and specificity for target antigens minimize adverse reactions and their molecular size results in extended circulation times relative to small molecule pharmaceuticals. The ability to customize the pharmacokinetics in a rational manner can enhance the potential for these and other classes of biologicals. We have systematically studied the effect of site-specific pegylation of the Fab‵ fragment of the anti-GPIIb/IIIa, αVβ3 antibody c7E3. Regardless of the molecular weight of the PEG molecules, the intrinsic affinity of the resulting constructs remained unchanged. However, in functional assays measuring inhibition of platelet aggregation, the calculated IC50 values of the conjugates decreased with increasing molecular weight of the conjugated PEG. It was determined that the molecular size of the conjugates affects antigen accessibility and whereas high levels of binding to antigen molecules on cells with high antigen density can be demonstrated with the Fab fragment, comparable levels are not achievable with large molecular weight conjugates. In spite of the inability of the larger PEG constructs to achieve saturation binding, functional inhibition of platelet aggregation consistent with saturation binding was demonstrated and the increased molecular size of the conjugates led to predictably prolonged inhibition of platelet aggregation.

In vitro setting of dose-effect relationships of 32 metal compounds in the Balb/3T3 cell line, as a basis for predicting their carcinogenic potential.

The results are reported of the second stage in a programme for a systematic in vitro study on the carcinogenic potential of metal compounds with Balb/3T3 clone A31-1-1 mouse fibroblasts. Nineteen metal compounds that exhibited a strong cytotoxic effect during a previous screening run with a 100 microM fixed dose were tested with a 72-hour exposure over a wide range of concentrations (from 0.1 microM to 1000 microM), to produce dose-effect curves to permit extrapolation of the 50% inhibition concentration (IC50) values for each metal compound. This allows the establishment of a suitable range of doses for individual metal species, for use in the subsequent Balb/3T3 assay based on a two-stage concurrent cytotoxicity and morphological transformation protocol. Another 13 metal compounds were also tested, to determine whether the Balb/3T3 cell transformation assay is really a valuable in vitro model in relation to the problem of metal speciation. Of the metal compounds assayed, 26 showed a dose related cytotoxic response with calculated IC50 values ranging from 0.25 microM (CH3HgCl) to 140 microM [(C5H5)2TiCl2], whereas six metal compounds, namely (NH4)6Mo7O24*4H2O, CH3AsO(OH)2, C2H6AsNaO2(3H2O, KBr, CrCl3*6H2O and (NH4)2[TiO(C2O4)2]*H2O, displayed no observable cytotoxicity or low cytotoxicity at all the doses tested. The determination of IC50 values permits a ranking of the cytotoxicity responses of metal compounds with the highest cytotoxicities. Dose-effect curves and IC50 values of different chemical forms of individual metal compounds of As, Br, Cr, Hg, Ir, Pt, Te, Ti and V (cationic/anionic inorganic or organometallic species) showed clearly how the chemical nature of the metal strongly influences the toxic response. This confirms that the Balb/3T3 cell line is a valuable in vitro model with respect to the problem of metal speciation. This is a fundamental aspect to be considered when incorporating the results from in vitro cell transformation assays of the carcinogenic potential of metal compounds into regulatory testing schemes. In this context, the choice of test metal species for the development and validation of such assays cannot disregard the possibility that humans will be exposed to specific chemical forms of individual metal compounds (different oxidation states, and inorganic or organometallic natures) that can profoundly affect their toxicity.

Enoyl-ACP Reductase (FabI) of Haemophilus influenzae: Steady-State Kinetic Mechanism and Inhibition by Triclosan and Hexachlorophene

Steady-state kinetics, equilibrium binding, and primary substrate kinetic isotope effect studies revealed that the reduction of crotonyl-CoA by NADH, catalyzed by Haemophilus influenzae enoyl-ACP reductase (FabI), follows a rapid equilibrium random kinetic mechanism with negative interaction among the substrates. Two biphenyl inhibitors, triclosan and hexachlorophene, were studied in the context of the kinetic mechanism. IC50 values for triclosan in the presence and absence of NAD+ were 0.1 ± 0.02 and 2.4 ± 0.02 μM, respectively, confirming previous observations that the E–NAD+ complex binds triclosan more tightly than the free enzyme. Preincubation of the enzyme with triclosan and NADH suggested that the E–NADH complex is the active triclosan binding species as well. These results were reinforced by measurement of binding kinetic transients. Intrinsic protein fluorescence changes induced by binding of 20 μM triclosan to E, E–NADH, E–NAD+, and E–crotonyl-CoA occur at rates of 0.0124 ± 0.001, 0.0663 ± 0.002, 0.412 ± 0.01, and 0.0069 ± 0.0001 s−1, respectively. The rate of binding decreased with increasing crotonyl-CoA concentrations in the E-crotonyl-CoA complex, and the extrapolated rate at zero concentration of crotonyl-CoA corresponded to the rate observed for the binding to the free enzyme. This suggests that triclosan and the acyl substrate share a common binding site. Hexachlorophene inhibition, on the other hand, was NAD+- and time-independent; and the calculated IC50 value was 2.5 ± 0.4 μM. Steady-state inhibition patterns did not allow the mode of inhibition to be unambiguously determined, but binding kinetics suggested that free enzyme, E–NAD+, and E–crotonyl-CoA have similar affinity for hexachlorophene, since the kobss were in the same range of 20–24 s−1. When the E–NADH complex was mixed with hexachlorophene ligand, concentration-independent fluorescence quenching at 480 nm was observed, suggesting at least partial competition between NADH and hexachlorophene for the same binding site. Mutual exclusivity studies, together with the above-discussed results, indicate that triclosan and hexachlorophene bind at different sites of H. influenzae FabI.

Development of a Homogeneous Time-Resolved Fluorescence Assay for High Throughput Screening to Identify Lck Inhibitors: Comparison with Scintillation Proximity Assay and Streptavidin-Coated Plate Assay

This study details the development of a homogeneous time-resolved fluorescence (HTRF) high throughput screening assay to identify inhibitors of Lck. HTRF was compared with scintillation proximity and streptavidin-coated plate assays. Because of the differences in the sensitivity of detection of phosphotyrosine among the three assays, different amounts of enzyme were used. However, the concentrations of the other assay components were standardized. When using similar assay conditions, the calculated IC50 values of inhibitory compounds were independent of assay format. Furthermore, filtration experiments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr peptide substrate was less than autophosphorylation of the Lck enzyme; this was due to the low Km value for biotinyl poly-Glu,Ala,Tyr. In the HTRF assay, small amounts of enzyme and high concentrations of ATP could be used, thereby minimizing the effects of autophosphorylation. Higher ATP concentration would also minimize the effect of ATP competitors. Using this technology, it may be possible to find novel kinase inhibitors that do not act at the ATP binding site of protein tyrosine kinases.

Comparison of AMP579 and adenosine in inhibition of cell-cell interaction between human neutrophil and vascular endothelial cell

The purpose of the present study was to compare inhibitory effects between AMP579 (a new adenosine analog) and adenosine (Ado) in attenuating an interaction between human neutrophils (PMNs) and cultured human umbilical vein endothelial cells (HUVECs). PMN activation was determined by superoxide anion (O2–) production and degranulation (myeloperoxidase release). Cell–cell interaction was quantitated by adherence of fluorescent labeled PMNs to HUVECs. AMP579 inhibited O2– (nM/5 × 106 PMN) from fMLP-activated human PMNs (55.3 ± 3.1) in a concentration-dependent manner ranging from 31.1 ± 2.9 at 10 nM to 11.7 ± 0.9 at 10 ± μM, all P < 0.01 vs. fMLP group. In the same dose range, however, Ado showed significant inhibition only at 1 μM (30.3 ± 4.1) and 10 ± μM (27.5 ± 4.3) vs. the fMLP group. The calculated IC50 value (0.11 ± 0.05 μM) in AMP579 group was significantly less than that in the Ado group (4.1 ± 1.2 μM). Although there was no group difference on PMN myeloperoxidase release (percent inhibition from fMLP) between AMP579 and Ado at concentrations greater than 1 μM (52.9 ± 5.2 vs. 46.4 ± 5.9), AMP579 showed significant attenuation of degranulation compared with Ado at 10 nM (31.7 ± 2.5 vs. 11.6 ± 1.9) and 100 nM (48.2 ± 4.6 vs. 25.6 ± 3.8), respectively, suggesting that AMP579 is more potent in inhibiting PMN activation. AMP579 reduced PMN adherence to TNFα-stimulated HUVEC (fluorescent units/well) in a concentration-dependent manner from 472 ± 32 at 10 nM to 214 ± 15 at 10 μM vs. 675 ± 54 in the TNFα group. At 10 nM and 100 nM, adenosine did not attenuate PMN adherence, while it showed significant inhibition at 1 (504 ± 45) and 10 μM (435 ± 50), respectively. The IC50 value (2.8 ± 1 μM) for AMP579 was significantly lower than that (41 ± 8 μM) in the Ado group. The results from the present study suggest that 1) AMP579 directly inhibits adherence-independent superoxide radical generation and degranulation from activated PMNs and attenuates cell–cell interaction between PMNs and vascular endothelial cells by preventing damage on endothelial cells. 2) AMP579 exerts more potent protective effect compared with adenosine at a lower dose range, indicating its prospect for clinical application. Drug Dev. Res. 49:266–272, 2000. © 2000 Wiley-Liss, Inc.

Different inhibitory actions of IGFBP-1, -2 and -4 on IGF-I effects in vascular smooth muscle cells.

IGF-I is involved in the regulation of metabolism, growth and migration of vascular smooth muscle cells (VSMCs). We have studied how IGFBP-1, -2 and -4 modulate IGF-I-induced DNA and protein synthesis in cultured rat VSMCs. DNA and protein synthesis were measured as incorporation of [3H]thymidine and [3H]leucine into DNA and protein respectively. Western immunoblot was used to detect IGFBPs in conditioned medium and solution hybridization was used to measure IGFBP gene expression. IGF-I stimulated DNA synthesis with an EC50 of 44 pM, reaching a maximal effect at 1 nM. An IGF-I concentration of 1 nM was subsequently used in the experiments with IGFBPs. IGFBP-1 and IGFBP-4 acted in an inhibitory manner on IGF-I-induced DNA synthesis with calculated IC50 values of 1.6 nM and 6.2 nM respectively. IGFBP-2 (16 nM) also inhibited the growth response to IGF-I, but this effect could only be obtained if the two peptides were pre-incubated together for 2 h prior to addition to the cells. IGFBP-1, -2 and -4 inhibited IGF-I-induced protein synthesis in a similar way. Immunoblot of the incubation medium showed little degradation of IGFBP-2 and -4 for up to 24 h. mRNA for IGFBP-2 and -4, but not for IGFBP-1 was detected in the VSMCs. Endogenous IGFBP-2 and -4 could be detected by immunoblot in the conditioned medium but only if it was concentrated. In conclusion, IGFBP-1, -2 and -4, of which IGFBP-2 and -4 may be locally derived, act as inhibitors with different potencies on IGF-I effects in VSMCs.

Uraemic guanidino compounds inhibit γ-aminobutyric acid-evoked whole cell currents in mouse spinal cord neurones

Guanidine, creatinine (CTN), methylguanidine (MG) and guanidinosuccinic acid (GSA) are four endogenous guanidino compounds with proven neuroexcitatory actions, and putative pathophysiological significance as uraemic toxins. The effects of these uraemic guanidino compounds, were studied on whole-cell current evoked by γ-amino butyric acid (GABA) on mouse spinal cord neurones in vitro. CTN, MG and GSA concentration dependently blocked GABA-evoked current with calculated IC50 values (± SE) of 9.6±0.9, 9.7±1.5 and 5.1±0.4 mM, respectively. CTN, MG and GSA were shown to block inward and outward currents to the same extent, demonstrating voltage independent block of GABA-evoked current by these compounds. Guanidine, however, evoked inward whole-cell currents, which were almost completely blocked by strychnine, indicating that the guanidine-evoked current might have been due to glycine receptor activation.

Effects of competitive NMDA receptor antagonists on excitatory amino acid-evoked currents in mouse spinal cord neurones.

The effects of CGP 37849 [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoate] and its ethylester CGP 39551 on whole-cell currents evoked by the endogenous excitatory amino acids, L-glutamate and L-aspartate, were studied in cultured mouse spinal cord neurones. Although CGP 37849 was the more potent compound, both antagonists inhibited 20 microM L-aspartate or 2 microM L-glutamate currents concentration-dependently and reversibly. We calculated IC50 values of 370 +/- 180 nM for CGP 37849 and 2200 +/- 140 nM for CGP 39551 (inhibition of L-aspartate current), and 210 +/- 25 nM for CGP 37849 and 6000 +/- 4700 nM for CGP 39551 (inhibition of L-glutamate current). Both CGP 37849 and CGP 39551 selectively blocked N-methyl-D-aspartate (NMDA)-evoked inward current. Current evoked by 5 microM kainate or 5 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was unaffected by 10 microM CGP 39551. Current evoked by NMDA was concentration-dependently blocked by CGP 39551 with an IC50 of 2100 +/- 220 nM. After application of 10 microM CGP 37849, 17 +/- 6% of the current evoked by 5 microM L-glutamate remained. This residual current was due to non-NMDA receptor activation since application of 25 microM 2-amino-5-phosphonovalerate (APV) together with CGP 37849 did not significantly alter the residual current, whereas application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) with CGP 37849 did significantly inhibit this current. Clamping cells at potentials ranging from -80 to +60 mV showed a linear potential--current relationship for the 20 microM L-aspartate-evoked current with reversal potential around 0 mV. The proportion of the L-aspartate current antagonized by CGP 37849 or CGP 39551 appeared to be independent of clamping potential. The concentration--current relationship of L-aspartate in the absence of the antagonists showed an EC50 of 49 +/- 14 microM. Upon application of 1 microM CGP 37849 and 10 microM CGP 39551, the L-aspartate concentration--current curve shifted to higher concentrations, and resulted in a 5- and 13-fold increase in the EC50 of L-aspartate, respectively, whereas Imax was not changed by application of the antagonists. Thus, the potent NMDA antagonists CGP 37849 and CGP 39551 were shown to inhibit excitatory amino acid responses specifically by competitive binding to the neurotransmitter recognition site of the NMDA receptor. Selective, competitive antagonism of L-glutamate- and L-aspartate-evoked NMDA receptor responses probably underlies the effects of CGP 37849 and CGP 39551 such as their anticonvulsant, neuroprotectant and antidepressant actions.

Lead and catechol hematotoxicity in vitro using human and murine hematopoietic progenitor cells.

In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 micromol/L) than for human cord blood cells (IC50 = 4 micromol/L). Lead was 10-15 times more toxic to human hematopoietic cells (IC50 = 61 micromol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 micromol/L; B6C3F1, IC50 = 536 micromol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 micromol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 micromol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations.