Enhancement of NMDA-mediated responses by cyanide

The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg²⁺ resulted in a five-fold increase in NMDA-induced peak of [Ca²⁺]_i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg²⁺. Cyanide enhanced the immediate rise in [Ca²⁺]_i produced by NMDA, followed over a 1–5 min period by a gradual increase of [Ca²⁺]_i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg²⁺. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [³H]MK-801 to rat forebrain membranes in the presence of Mg²⁺, whereas in the absence of Mg²⁺, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca²⁺ influx and inward current by interacting with the Mg²⁺ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg²⁺ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca²⁺ influx resulting from cellular energy reserve depletion.Abbreviations NMDA N-Methyl-D-Aspartate - EAA excitatory amino acid - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohept-5,10-imine maleate     

Synthesis, radiolabelling and biological characterization of (D)-7-iodo-N-(1-phosphonoethyl)-5-aminomethylquinoxaline-2,3-dione, a glycine-binding site antagonist of NMDA receptors

(D)-7-Iodo-N-(1-phosphonoethyl)-5-aminomethylquinoxaline-2,3 -dione (I-PAMQX), is a potent, in vivo active antagonist acting at the glycine binding site of the NMDA receptor complex. Radioiodinated [131I]I-PAMQX was prepared with good yields and high specific activity from its 7-bromo analogue. Biodistribution studies of [131I]I-PAMQX in mice showed a relatively slow clearance from the blood. The uptake of radioactivity was highest in the kidneys, moderate in the heart, lung, liver and bones, and low in the brain.     

Activation of human IK and SK Ca2+ -activated K+ channels by NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime)

We have identified and characterized the compound NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) as a potent activator of human Ca2+ -activated K+ channels of SK and IK types, whereas it is devoid of effect on BK type channels. IK- and SK-channels have previously been reported to be activated by the benzimidazolinone, 1-EBIO and more potently by its dichloronated-analogue, DC-EBIO. NS309 is at least 1000 times more potent than 1-EBIO and at least 30 times more potent than DC-EBIO when the compounds are compared on the same cell.     

Novel indoline-2,3-dione derivatives as inhibitors of aminopeptidase N (APN)

Aminopeptidase N (APN/CD13), as a zinc-containing ectoenzyme, plays a critical role in the process of tumor angiogenesis, invasion and metastasis. Through the docking-based virtual screening of chemical databases and the further activity assay, we discovered that compound 10c exhibits potent and selective inhibitory ability towards APN. In addition, a series of indoline-2,3-dione derivates have been designed and synthesized as APN inhibitors. The results of preliminary activity evaluation showed that compound 12a (IC₅₀ = 0.074 ± 0.0026 μM) exhibited the best inhibitory activity against APN, which could be used for further anticancer agent research.     

A specific, photolabile and irreversible antagonist (L662,025) of the PAF-receptor

PAF (0.2 microM) induced maximal platelet aggregation in human PRP and [3H]-PAF (1-5 nM) binding to platelet membrane preparations had Kd value of 3.8 nM and Bmax of 200 fmoles/mg of protein. Without UV irradiation, a synthetic azido tetrahydrofuran derivative L662,025 was a reversible and competitive PAF-receptor antagonist with IC50 values of 5.6 +/- 0.3 microM (platelet aggregation) and 1.0 +/- 0.25 microM (receptor binding). Photolysis of L662,025 in the presence of PRP produced an irreversible inhibition of platelet aggregation and specific binding of [3H]-PAF (1 nM). L662,025 did not affect collagen- or ADP-induced human platelet aggregation before or after photolysis. It is a new probe that can be used to identify and characterize the PAF-receptor.     

Inhibition of transmembrane movement and metabolism of platelet activating factor (PAF-acether) by a specific antagonist, BN 52021

Incorporation of 1-[3H]-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H] PAF-acether) into rabbit platelet phosphatidylcholine (PC) was inhibited by a specific antagonist, BN 52021 (IC50 5.6 X 10(-6) M, maximal effect, i.e 70% inhibition, at 10(-4) M). Under the same conditions, [3H] lyso-PAF-acether incorporation remained 9 fold lower, compared to PAF-acether, without any effect of BN 52021. Upon cell lysis, both phospholipids attained the same rate of metabolic conversion, corresponding to a 1.15-fold and a 12-fold increase for PAF-acether and lyso-PAF-acether, respectively. In none of these cases was BN 52021 effective. It is concluded that transmembrane movement of the two phospholipids represents the limiting step of their metabolism. The higher rate of PAF-acether conversion by intact platelets could involve its binding to a membrane receptor, as suggested by the inhibitory effect of BN 52021, the significance of which is discussed.     

CV-3988 - a specific antagonist of platelet activating factor (PAF)

CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate was shown to be a specific inhibitor of platelet activating factor (PAF). This compound in concentrations of 3 x 10(-6) to 3 x 10(-5)M inhibited aggregation of rabbit platelets induced by PAF (3 x 10(-8)M), while it had no effect on the aggregation induced by arachidonic acid, ADP, collagen or A-23187. CV-3988 alone even at a concentration of 10(-3)M had no effect on platelet aggregation. The inhibitory action of CV-3988 on the PAF-induced aggregation was independent of the formation of micelles. The PAF (0.1 to 1.0 micrograms/kg, i.v.)-induced hypotension in anesthetized rats was also inhibited dose-dependently by the i.v. administration of CV-3988 (1 and 10 mg/kg), while the hypotensive actions induced by the i.v. administration of acetylcholine (1 micrograms/kg), arachidonic acid (1 mg/kg), bradykinin (10 micrograms/kg), isoproterenol (1 microgram/kg) and histamine (100 micrograms/kg) were not altered by CV-3988 (10 mg/kg, i.v.). All these findings indicate that CV-3988 specifically inhibits the action of PAF in vitro and in vivo. This is the first report of a PAF antagonist which can specifically inhibit the PAF-induced hypotension as well as the PAF-induced platelet aggregation.     

3-(Aminomethyl)piperazine-2,5-dione as a novel NMDA glycine site inhibitor from the chemical universe database GDB

Docking of randomly selected compounds from the chemical universe database GDB-11, which contains all organic molecules up to 11 atoms of C, N, O, F possible under consideration of simple chemical stability and synthetic feasibility rules, into the NMDA receptor glycine site (1pb7.pdb) lead to the identification of 3-(aminomethyl)piperazine-2,5-dione 3 and its close analog 5-(aminomethyl)piperazine-2,3-dione 4 as possible new ligands for this drug target, which is implicated in synaptic plasticity, neuronal development, learning and memory. Synthesis of these compounds in 4 and 6 steps, respectively, and testing by radioligand displacement assays and electrophysiological measurements in Xenopus oocytes show that while 4 is inactive, 3 is indeed an inhibitor of glycine, with an estimated K_D of 50 μM.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

Prolonged inhibition of pressor response to vasopressin by a potent specific antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid) 2-(O-methyl)tyrosine]arginine-vasopressin

The specificity, the potency, and the duration of action of [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid) 2-(O-methyl)tyrosine]arginine-vasopressin[d(CH2)5Tyr(Me)AVP] to antagonize pressor responses to arginine vasopressin (AVP) was examined in pentobarbital-anaesthetized rats. Injection of the compound (4 micrograms.kg-1 i.v.) prevented pressor responses to i.v. infusions of supramaximal doses of AVP, but not to i.v. infusions of another peptide, angiotensin II (Ag II). The antagonism of AVP persisted for at least 3 h. Since i.v. injection of the compound in the absence of exogenous administration of AVP did not cause any change in the arterial pressure of rats, it appears that the compound is devoid of agonistic pressor activity. The results show that d(CH2)5Tyr(Me)AVP is a potent and a specific antagonist of pressor responses to AVP.     

Dehydroepiandrosterone activates endothelial cell nitric-oxide synthase by a specific plasma membrane receptor coupled to Galpha(i2,3)

The adrenal steroid dehydroepiandrosterone (DHEA) has no known cellular receptor or unifying mechanism of action, despite evidence suggesting beneficial vascular effects in humans. Based on previous data from our laboratory, we hypothesized that DHEA binds to specific cell-surface receptors to activate intracellular G-proteins and endothelial nitric-oxide synthase (eNOS). We now pharmacologically characterize a putative plasma membrane DHEA receptor and define its associated G-proteins. The [3H]DHEA binding to isolated plasma membranes from bovine aortic endothelial cells was of high affinity (K(d) = 48.7 pm) and saturable (B(max) = 500 fmol/mg protein). Structurally related steroids failed to compete with DHEA for binding. The putative DHEA receptor was functionally coupled to G-proteins, because guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) inhibited [3H]DHEA binding to plasma membranes by 69%, and DHEA increased [35S]GTPgammaS binding by 157%. DHEA stimulated [35S]GTPgammaS binding to Galpha(i2) and Galpha(i3), but not to Galpha(i1) or Galpha(o). Pretreatment of plasma membranes with antibody to Galpha(i2) or Galpha(i3), but not to Galpha(i1), inhibited the DHEA activation of eNOS. Thus, DHEA receptors are expressed on endothelial cell plasma membranes and are coupled to eNOS activity through Galpha(i2) and Galpha(i3). These novel findings should allow us to isolate the putative receptor and reevaluate the physiological role of DHEA activity.     

Synthesis and bioluminescence-inducing properties of autoinducer (S)-4,5-dihydroxypentane-2,3-dione and its enantiomer

The autoinducer (4S)-4,5-dihydroxypentane-2,3-dione ((S)-DPD, AI-2) facilitates chemical communication, termed ‘quorum sensing’, amongst a wide range of bacteria, The synthesis of (S)-DPD is challenging in part due to its instability. Herein we report a novel synthesis of (S)-DPD via (2S)-2,3-O-isopropylidene glyceraldehyde, through Wittig, dihydroxylation and oxidation reactions, with a complimentary asymmetric synthesis to a key precursor. Its enantiomer (R)-DPD, was prepared from d-mannitol via (2R)-2,3-O-isopropylideneglyceraldehyde. The synthesized enantiomers of DPD have AI-2 bioluminescence-inducing properties in the Vibrio harveyi BB170 strain.     

A glycine site‐specific NMDA receptor antagonist protects retina ganglion cells from ischemic injury by modulating apoptotic cascades

Glutamate neurotoxicity is one of the causative factors leading to neural degeneration including retina. Inhibition of NMDA receptors has been shown neuroprotective effects. However, specifically inhibition of glycine subunit in NMDA receptors and its effects on retina neural protection has not been tested. In this study, using a glycine site‐specific NMDA receptor antagonist, we investigated its neuroprotective effects on rat retinal ganglion cells (RGCs) from a transient ischemic injury and its possible underlying mechanisms. Following an ischemia/reperfusion injury the structural damages of rat retinas were assessed by an immunofluorescence method and the apoptosis of retinal neural cells was evaluated by using a terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) method. The survived RGCs were labeled by retrograde manner and counted on whole‐mounted retinas. In the presence of glycine site‐specific NMDA receptor antagonist, the thickness of retina was sustained, especially in the inner nuclear layers compared with mock controls. While a significantly higher numbers of TUNEL‐positive apoptotic cells and fewer of RGCs were observed in the retina without the glycine antagonist, indicating its strong protective roles. Some apoptotic factors such as Bax, Bcl‐2, CAMK II, COX1, COX4, Caspase‐3, and GRIN1 gene have been tested from retinal samples with or without the glycine antagonist. A significantly lower of expressions of Bax, CAMK II, COX1, COX4, Caspase‐3, and GRIN1 have been shown in the retinas with the antagonist. Bcl‐2/Bax ratio was significantly higher with the antagonist, suggested that the glycine site‐specific NMDA receptor antagonist protecting RGC death might through inhibition of apoptotic signaling. J. Cell. Physiol. 223:819–826, 2010. © 2010 Wiley‐Liss, Inc.     

Enzymatic adenylation of 2,3-dihydroxybenzoate is enhanced by a protein-protein interaction between Escherichia coli 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (EntA) and 2,3-dihydroxybenzoate-AMP ligase (EntE)

The Escherichia coli siderophore enterobactin is synthesized in response to iron starvation. 2,3-Dihydro-2,3-dihydroxybenzoate dehydrogenase (EntA) produces 2,3-dihydroxybenzoate (DHB), a biosynthetic intermediate. 2,3-Dihydroxybenzoate-AMP ligase (EntE) adenylates DHB, activating it for attachment to the NRPS substrate holo-EntB. Using analytical ultracentrifugation, we found that EntA undergoes concentration-dependent dimer-tetramer self-association (K(D) = 12.3 μM). We further found that EntA can form a specific complex with EntE. Pull-down assays revealed that recombinant EntA bait pulled down EntE from E. coli lysates, whereas recombinant EntE bait could pull down EntA. Addition of the SMCC cross-linker to a mixture of EntA and EntE resulted in a cross-linked product with a molecular mass of >250 kDa, suggesting a complex stoichiometry of one EntA tetramer and four EntE monomers. The effect of EntA on EntE activity was also examined. Addition of a 4-fold excess of EntA to an EntE assay mixture resulted in a 6-fold stimulation of EntE activity. EntA was also found to perturb the FRET signal between EntE donor residues and EntE-bound DHB. By following the EntA-dependent decrease in the magnitude of the EntE-DHB FRET signal, EntA-EntE binding behavior was found to be sigmoidal, suggesting the presence of both low- and high-affinity binding sites. The EntA-EntE interaction was also directly measured by isothermal titration calorimetry at 10 °C. The resulting binding isotherm fit well to a model describing two binding sites, supporting our AUC and fluorescence data. Taken together, our data show that tetrameric EntA optimally interacts with EntE, resulting in an enhancement of EntE activity.     

In vitro metabolism of (6,7-H3)estrone and of (6,7-H3) estradiol by the liver in pregnant guinea pigs]

Slices of pregnant guinea pig liver were incubated with (6,7-3H)estrone and with (6,7-3H)estradiol. Free, glucuro- and sulfo-conjugated fractions were isolated by specific extraction and hydrolysis. The radioactivity distribution in these 3 fractions demonstrated a predominance of conjugated compounds (95% of isolated estrogens) with slightly more glucuro-conjugated than sulfo-conjugated compounds. After isolating estrogens by TLC, we were able to determine estrone and estradiol in these 3 fractions from incubations with 3H-estrone or with 3H-estradiol by means of specific activity recrystallisation. Estriol was determined in glucuro-and sulfo-conjugated fractions after incubation with 3H-estrone as well as in sulfo-conjugated fraction after incubation with 3H-estradiol. Glucuro- or sulfo-conjugated estrone was the predominant estrogen after incubation with 3H-estrone just as after incubation with 3H-estradiol. This led us to conclude to an important 17beta-hydroxysteroid-dehydrogenase activity. The 16alpha-hydroxylastic-activity is weaker since estriol represented only 1,43 % of estrogens isolated after incubation with 3H-estrone and 0.82% after incubation with 3H-estradiol.     

Isatin (indole-2,3-dione) in urine and tissues: Detection and determination by gas chromatography—mass spectrometry

A simple procedure based upon capillary column gas chromatography-mass spectrometry (GC—MS) is described for the detection and determination of isatin (indole-2,3-dione) in body fluids and tissues. After addition of 5-methylisatin as internal standard to urine or tissue homogenates, organic extracts are dried and derivatized successively with hydroxylamine hydrochloride and the reagent N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). The tert.-butyldimethylsilyl derivatives obtained show good GC—MS properties and allow quantification by selected-ion monitoring of m/z 333 (isatin) and m/z 347 (internal standard). Adult and newborn human urine output values lie in the ranges 0.4–3.2 mg/mmol of creatinine (5–30 mg per 24 h) and 0.002–0.518 mg/mmol of creatinine, respectively. There is a discontinuous regional distribution in rat tissues. The GC—MS properties of a number of derivatives formed by successive reaction of isatin with hydroxylamine hydrochloride (or methoxyaminehydrochloride or ethoxyamine hydrochloride) and MTBSTFA, bis(trimethylsilyl)trifluoroacetamide, pentafluoropropionic anhydride or pentafluorobenzyl bromide are also described.     

Different roles of GABA and glycine in the modulation of chemosensory responses in Hydra vulgaris (Cnidaria,Hydrozoa)

Phylogenetic studies suggest that GABA and glycine receptors derive, as a result of divergent evolution, from a common ancestral protoreceptor originated in a unicellular organism. This raises the possibility that members of the ligand-gated ion channels (LGIC) superfamily might be widely present in living organisms including bacteria and primitive invertebrates. High-affinity GABA receptors occur in the tissues of Hydra vulgaris whose pharmacological characteristics compare with those of mammalian ionotropic GABA receptors. Behavioural studies have shown that activation of these GABA _A -like receptors by their allosteric modulators increases the duration of response to reduced glutathione (GSH). Recently, strychnine-sensitive glycine receptors have been shown to occur in Hydra tissues. Activation of these glyR also results in increased duration of the response to GSH. In order to investigate the contribution of endogenous transmitters to the modulation of the feeding response, we studied the effects of exposing the polyps to brief depolarizing pulses prior to the GSH test. A severe inhibition of the response was observed following exposure to KCl or veratridine. Administration of GABA or muscimol counteracted the effects of the pulses in a dose-dependent manner. The effects of GABA or muscimol were suppressed by the GABA _A -specific antagonist gabazine both in pulse-untreated and treated polyps. By contrast, glycine and its agonist taurine were not able to restore the physiological duration of response in pulse-treated Hydra, while another glyR agonist, β-alanine, partially reduced the pulse-induced inhibition. We conclude that GABA appears to be the major inhibitory transmitter responsible for the regulation of the feeding response. Molecular studies aimed at identifying GABA receptor subunits are in progress.     

The specific targeting of immune regulation: T-cell responses against Indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in many settings including cancer. In recent years, we have described spontaneous CD8(+) as well as CD4(+) T-cell reactivity against IDO in the tumor microenvironment of different cancer patients as well as in the peripheral blood of both cancer patients and to a lesser extent in healthy donors. We have demonstrated that IDO-reactive CD8(+) T cells were peptide-specific, cytotoxic effector cells, which are able to recognize and kill IDO-expressing cells including tumor cells as well as dendritic cells. Consequently, IDO may serve as a widely applicable target for immunotherapeutic strategies with a completely different function as well as expression pattern compared to previously described antigens. IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, and IDO-based immunotherapy may consequently be synergistic with additional immunotherapy. In this regard, we have shown that the presence of IDO-specific T cells boosted immunity against CMV and tumor antigens by eliminating IDO(+) suppressive cells and changing the regulatory microenvironment. The current review summarizes current knowledge of IDO as a T-cell antigen, reports the initial results that are suggesting a general function of IDO-specific T cells in immunoregulation, and discusses future opportunities.     

Enzymatic and kinetic properties of blood coagulation factor XIIIa and guinea pig liver transglutaminase utilizing (6-[N-(4-aminobutyl)-N-ethylamino]-2,3-dihydrophthalazine-1,4-dione,as a novel,specific and sensitive chemiluminescent substrate

A novel and sensitive chemiluminescent assay is described to quantitate the acyl transfer activities of blood coagulation factor XIIIa or liver transglutaminase using aminobutyl-N-ethyl-isoluminol as acyl acceptor and N,N′-dimethylcasein, human plasma fibrinogen or fibronectin as acyl donors. The method involved covalently linking aminobutyl-N-ethyl-isoluminol through its free amino group with the γ-carboxamide of protein-bound glutamine resulting in an isopeptide bond; a reaction catalysed by both transglutaminase and factor XIIIa. The protein-bound aminobutyl-N-ethyl-isoluminol was separated from non-conjugated amine by precipitation with trichloroacetic acid. The protein–amine conjugate was dissolved in 500 mmol/L NaOH, oxidized using 15 mmol/L ammonium persulphate and light emission quantitated using a luminometer. Optimal conditions were established to detect factor XIIIa and transglutaminase activities with the chemiluminescent assay. Specificity was demonstrated by lack of activity in the presence of ethylenediamine tetra-acetic acid or unactivated factor XIII, or boiled enzymes, and by competitive inhibition with putrescine and 5′-(biotinamido) pentylamine. The enzymatic and kinetic properties of factor XIIIa and transglutaminase in utilizing aminobutyl-N-ethyl-isoluminol as an acyl acceptor substrate were comprehensively documented. The reaction could be carried out in either a purified system or a complex plasma or cell lysates milieu. The assay is sensitive, specific, and eliminates a need for radioactive reagents. The assay could be used to photolabel reactive glutamines in substrates. The assay could also be adapted to a variety of solid- and solution-phase formats and is amenable to X-ray film and/or light photography imaging. © 1998 John Wiley & Sons, Ltd.