Protein tyrosine kinase activity as a prognostic parameter in breast cancer, a pilot study

The interaction of omega (benzodiazepine) modulatory drugs with transiently expressed alpha 1 beta 2 gamma 2 and alpha 5 beta 2 gamma 2 forms of the rat GABAA receptor was investigated using [3H]flumazenil as a probe in in vitro radioligand binding assays. The imidazopyridines alpidem and zolpidem exhibited pronounced selectivity for the alpha 1- compared to the alpha 5-containing construct, whereas omega (benzodiazepine) site modulatory compounds from other chemical series including diazepam, tetrazepam, zopiclone, triazolam, bretazenil and midazolam behaved as relatively non-selective drugs. In the presence of 10 microM gamma-aminobutyric acid (GABA) the potencies of diazepam, flunitrazepam and midazolam to inhibit [3H]flumazenil binding to the alpha 1-construct were increased 3 to 5 fold, whereas with 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester a 2.5-fold reduction in potency was observed. Similar modulatory effects of GABA were obtained with these drugs, using the alpha 5-construct. We suggest that these GABA shift determinations of [3H]flumazenil binding can be used as a rapid test to evaluate the intrinsic activities of omega modulatory compounds.     

Theoretical Analysis on Geometry Structure and Electronic Structure of LaNi4Co

Influence of Y~(3 ) on Structure and Electrochemical Property of LiMn_2O_4     

Theoretical Analysis on Geometry Structure and Electronic Structure of LaNi_4Co

Theoretical Analysis on Geometry Structure and Electronic Structure of LaNi_4Co     

Electronic structure calculations of rare-earth intermetallic compound YAg using ab initio methods

The structural, elastic and electronic properties of YAg-B2(CsCl) were investigated using the first-principles calculations. The energy band structure and the density of states were studied in detail, including partial density of states (PDOS), in order to identify the character of each band. The structural parameters (lattice constant, bulk modulus, pressure derivative of bulk modulus) and elastic constants were also obtained. The results were consistent with the experimental data available in the literature, as well as other theoretical results.     

Structural, electronic and elastic properties of YCu from first principles

The structural, electronic and elastic properties of YCu compound in the B2 (CsCl) phase were investigated using the density functional theory (DFT) within the generalized gradient approximation (GGA). The electronic density of states (DOS) obtained in this way accorded well with the results of a recent study utilizing the full-potential linearized augmented plane wave (FLAPW) method. We also found that the density of d-states at the Fermi energy was low. The calculated equilibrium properties such as lattice constant, bulk modulus and its first derivative, and the elastic constants were in good agreement with experimental and theoretical results.     

Successful polymerase chain reaction-based diagnosis of fungal meningitis in a patient with chronic granulomatous disease

The structural and electronic properties of seventeen alkylxanthine derivatives were calculated using the MO program PM3 to elucidate the key features related to their inhibitory activity on phosphodiesterase (PDE) IV isoenzyme. Except for 7-alkylxanthine derivatives, a good correlation could be established between the distance between the tops of the two alkyl groups at the N1 and N3 positions of the xanthine skeleton (molecular length) and the PDE IV inhibitory activity (r=0.973, n=13). The same inhibitory activity could also be significantly correlated with the following electronic parameters of alkylxanthines: HOMO energy (r=0.850), absolute hardness (r=-0.806), and absolute electronegativity (r=-0.825). These results suggest that the electronic properties are partly responsible for PDE IV inhibition as far as the effects of structural properties associated with molecular length are concerned. Alkylxanthines may also act as electron donors in the charge-transfer interaction with the active sites on PDE IV isoenzyme.     

Topology of ligand binding sites on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of both alpha subunits there exist the binding sites for agonists such as the neurotransmitter acetylcholine (ACh) and for competitive antagonists such as d-tubocurarine. Agonists trigger the channel opening upon binding while competitive antagonists compete for the former ones and inhibit its pharmacological action. Identification of all residues involved in recognition and binding of agonist and competitive antagonists is a primary objective in order to understand which structural components are related to the physiological function of the AChR. The picture for the localisation of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are mainly located on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are sequentially identical, the observed high and low affinity for agonists on the receptor is conditioned by the interaction of the alpha subunit with the delta or the gamma chain, respectively. This relationship is opposite for curare-related drugs. This molecular interaction takes place probably at the interface formed by the different subunits. The principal component for the agonist/competitive antagonist binding sites involves several aromatic residues, in addition to the cysteine pair at 192-193, in three loops-forming binding domains (loops A-C). Other residues such as the negatively changed aspartates and glutamates (loop D), Thr or Tyr (loop E), and Trp (loop F) from non-alpha subunits were also found to form the complementary component of the agonist/competitive antagonist binding sites. Neurotoxins such as alpha-, kappa-bungarotoxin and several alpha-conotoxins seem to partially overlap with the agonist/competitive antagonist binding sites at multiple point of contacts. The alpha subunits also carry the binding site for certain acetylcholinesterase inhibitors such as eserine and for the neurotransmitter 5-hydroxytryptamine which activate the receptor without interacting with the classical agonist binding sites. The link between specific subunits by means of the binding of ACh molecules might play a pivotal role in the relative shift among receptor subunits. This conformational change would allow for the opening of the intrinsic receptor cation channel transducting the external chemical signal elicited by the agonist into membrane depolarisation. The ion flux activity can be inhibited by non-competitive inhibitors (NCIs). For this kind of drugs, a population of low-affinity binding sites has been found at the lipid-protein interface of the AChR. In addition, several high-affinity binding sites have been found to be located at different rings on the M2 transmembrane domain, namely luminal binding sites. In this regard, the serine ring is the locus for exogenous NCIs such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222, phencyclidine, and trifluoromethyliodophenyldiazirine. Trifluoromethyliodophenyldiazirine also binds to the valine ring, which is the postulated site for cembranoids. Additionally, the local anaesthetic meproadifen binding site seems to be located at the outer or extracellular ring. Interestingly, the M2 domain is also the locus for endogenous NCIs such as the neuropeptide substance P and the neurotransmitter 5-hydroxytryptamine. In contrast with this fact, experimental evidence supports the hypothesis for the existence of other NCI high-affinity binding sites located not at the channel lumen but at non-luminal binding domains. (ABSTRACT TRUNCATED)     

Comparative autoradiographic distribution of central omega (benzodiazepine) modulatory site subtypes with high, intermediate and low affinity for zolpidem and alpidem

Pharmacological characterization of [3H]benzodiazepine binding to membrane preparations of adult rat hippocampus and neonatal rat brain have demonstrated, in addition to the omega 1 and omega 2 populations of central omega benzodiazepine binding sites associated with GABAA receptors, the existence of binding sites with microM affinity for the imidazopyridines zolpidem and alpidem. In the present study we have investigated their comparative autoradiographic distribution using [3H]flumazenil as a ligand. In the neonatal rat CNS, the imidazopyridine derivatives zolpidem and alpidem were found to discriminate two [3H]flumazenil binding site populations with an IC50 value ratio of more than 200-fold. In the different regions investigated (spinal cord, striatum, neocortex and inferior colliculus) the low affinity component had IC50 values of 20-40 microM (zolpidem) and 5-15 microM (alpidem) and accounted for ca. 50% of the total binding site population. In the adult rat, these imidazopyridine derivatives displayed a greater displacing potency in the cerebellum (IC50 = 6 and 36 nM, respectively) than in the hippocampus (IC50 = 37 and 403 nM, respectively). In the cerebellum, [3H]flumazenil binding was fully displaced by 1 microM of either compound and Hill coefficients of displacement curves were close to unity. In the hippocampus, 25% of [3H]flumazenil binding were resistant to 3 microM zolpidem or 1 microM alpidem, but were displaced by 100 microM of either compound. CL 218,872 also displayed a greater displacing potency in the cerebellum (IC50 = 83 nM) than in the hippocampus (IC50 = 711 nM) but [3H]flumazenil binding in the hippocampus was fully displaced by 10 microM of this compound. In adult rat hippocampus, zolpidem and alpidem were found to discriminate between three central omega site subtypes which display high (IC50 = 31 and 6.1 nM, for these imidazopyridine derivatives. In contrast, CL 218,872 discriminated between omega 1 and omega 2 sites but not between two omega 2 receptor subpopulations. omega 1 sites were mainly localized in layer IV of the sensorimotor cortex, cerebellum, substantia nigra, olfactory bulb and inferior colliculus. omega 2I sites were present in the cortical mantle (with higher levels in the cingulate and olfactory than in the sensorimotor cortex) and in subcortical (hippocampus, hypothalamus and nucleus accumbens) limbic structures. In the hippocampus, hypothalamus, spinal cord and nucleus accumbens, omega 2L sites accounted for more than 25% of the specific [3H]flumazenil binding; the density of these sites was minor in the cortex and in most pyramidal and extrapyramidal system structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electronic and atomic-size effects on the omega phase formation in transition-metal based B.C.C. alloys

Two criteria for the omega phase formation in transition-metal based b.c.c. alloys have been proposed based on the electronic structure calculations and experiments on the omega phase. One is the electronic effect and the other is the atomic-size effect. The former effect is enhanced when the alloying transition element has more valence electrons than the mother metal. The latter effect is remarkable when the alloying transition element has a smaller atomic size than the mother metal. The importance of such a size effect has been confirmed from a series of experiments on the omega phase formation in ZrTi alloys. Following these criteria the compositional range for the omega phase to exist in various Ti and Zr alloys can be understood consistently. Further, the second-nearest-neighbour atomic interactions were shown to be large in metals and alloys where the omega phase is formed.     

Muscarinic acetylcholine receptors in Torpedo electric organ: effect of guanine nucleotides

The effect of guanine nucleotides on the binding properties of presynaptic muscarinic receptors has been studied in a membrane preparation from the electric organ of Torpedo marmorata by measuring the competitive displacement of the radiolabelled antagonist, [3H]quinuclidinyl benzilate, by nonradioactive muscarinic ligands. The binding of the antagonists, atropine, scopolamine and pirenzepine was to a single class of sites [slope factors (pseudo Hill coefficients) close to 1] and was unaffected by 0.1 mM GTP. The binding of the N-methylated antagonists, N-methylatropine and N-methyl-scopolamine was more complex (slope factors less than 1) but also insensitive (N-methylatropine) to 0.1 mM GTP. Agonist binding was complex and could be resolved into two binding sites with relatively high and low affinities. The proportion of high-affinity sites varied with the nature of the agonist (15-80%). Agonist binding was depressed by 0.1 mM GTP, and the order of sensitivity was oxotremorine-M greater than carbamoylcholine greater than muscarine greater than acetylcholine greater than arecoline greater than oxotremorine. The binding of pilocarpine, a partial agonist, was unaffected by GTP. With carbamoylcholine as a test ligand the GTP effect on agonist binding was half-maximal at 12 microM. GDP and guanylylimidodiphosphate produced comparable inhibition of carbamoylcholine binding, but GMP and cyclic GMP were ineffective, as were various adenine nucleotides. Analysis of agonist binding in terms of a two-site model indicates that the predominant effect of guanine nucleotides is to reduce the number of sites of higher affinity.     

AMPA receptors and bacterial periplasmic amino acid-binding proteins share the ionic mechanism of ligand recognition

In order to identify key structural determinants for ligand recognition, we subjected the ligand-binding domain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor GluR-D subunit to site-directed mutagenesis. Based on the analysis of the [3H]AMPA-binding properties of the mutated binding sites, we constructed a revised three-dimensional model of the ligand-binding site, different in many respects from previously published models. In particular, our results indicate that the residues Arg507 and Glu727 represent the structural and functional correlates of Arg77 and Asp161 in the homologous bacterial lysine/ornithine/arginine-binding protein and histidine-binding protein, and directly interact with the alpha-carboxyl and alpha-amino group of the bound ligand, respectively. In contrast, Glu424, implicated previously in ionic interactions with the alpha-amino group of the agonist, is unlikely to have such a role in ligand binding. Our results indicate that glutamate receptors share with the bacterial polar amino acid-binding proteins the fundamental mechanism of amino acid recognition.     

Transmembrane residues of the parathyroid hormone (PTH)/PTH-related peptide receptor that specifically affect binding and signaling by agonist ligands

Polar residues within the transmembrane domains (TMs) of G protein-coupled receptors have been implicated to be important determinants of receptor function. We have identified mutations at two polar sites in the TM regions of the rat parathyroid hormone (PTH)/PTH-related peptide receptor, Arg-233 in TM 2 and Gln-451 in TM 7, that caused 17-200-fold reductions in the binding affinity of the agonist peptide PTH-(1-34) without affecting the binding affinity of the antagonist/partial agonist PTH-(3-34). When mutations at the TM 2 and TM 7 sites were combined, binding affinity for PTH-(1-34) was restored to nearly that of the wild type receptor. The double mutant receptors, however, were completely defective in signaling cAMP or inositol phosphate production in response to PTH-(1-34) agonist ligand. The results demonstrate that Arg-233 and Gln-451 have important roles in determining agonist binding affinity and transmembrane signaling. Furthermore, the finding that residues in TM 2 and TM 7 are functionally linked suggests that the TM domain topology of the PTH/PTH-related peptide receptor may resemble that of receptors in the rhodopsin/beta-adrenergic receptor family, for which structural and mutagenesis data suggest interactions between TMs 2 and 7.     

Effects of chronic U50,488H treatment on binding and mechanical responses of the rat hearts

The effects of chronic injection of U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamidel++ +), a selective kappa opioid agonist, on the properties of the binding sites of tritiated U69593 [(5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl)benzeneacetamide], another selective kappa opioid agonist, and mechanical responses to U50,488H of the heart were studied. Rats received injection twice a day with U50,488H for 4 days. Binding studies on the crude membrane homogenates revealed that there was no change in maximum binding, but a significant increase in Kd after the treatment, indicating that the number of kappa binding sites remained unchanged whereas the affinity of the binding sites to kappa-agonist decreased. The study on the mechanical responses to U50,488H in the isolated perfused heart preparation showed that although the agonist at 10(-6) M caused MR2266 reversible reductions in heart rate and force of contraction as well as ventricular ectopic beat in the heart of rats in the control group, its effects were absent in the U50,488H-treated group, indicating the development of tolerance to the mechanical effects of U50,488H on the heart. The results indicate that the development of tolerance to the mechanical effects of a kappa-agonist after chronic treatment with the agonist was not accompanied by down-regulation, but only a slight and significant reduction in affinity of kappa binding sites in the rat heart.     

Simple models for the omega phase transformation

In the late sixties, it was recognized that the omega phase transformation occurring in bcc Ti, Zr, and Hf alloys was a displacive transition which could be described, at least qualitatively, by simple models. Since the displacive wave responsible for the transition to perfect omega was a Brillouin zone boundary wave, displacements and volume changes were rather small, in contrast to the classical martensite case. The nonideal, or modulated omega phase requires further consideration, and calls for soliton models, for example. Recent first principles electronic structure calculations, briefly reported upon here, have also shed light on the athermal omega transformation. This paper is based on a presentation made in the symposium “Pre-transformation Behavior Related to Displacive Transformations in Alloys≓ presented at the 1986 annual AIME meeting in New Orleans, March 2-6, 1986, under the auspices of the ASM-MSD Structures Committee.     

Sequence properties of GPI-anchored proteins near the omega-site: constraints for the polypeptide binding site of the putative transamidase

Glycosylphosphatidylinositol (GPI) anchoring is a common post-translational modification of extracellular eukaryotic proteins. Attachment of the GPI moiety to the carboxyl terminus (omega-site) of the polypeptide occurs after proteolytic cleavage of a C-terminal propeptide. In this work, the sequence pattern for GPI-modification was analyzed in terms of physical amino acid properties based on a database analysis of annotated proprotein sequences. In addition to a refinement of previously described sequence signals, we report conserved sequence properties in the regions omega - 11...omega - 1 and omega + 4...omega + 5. We present statistical evidence for volume-compensating residue exchanges with respect to the positions omega - 1...omega + 2. Differences between protozoan and metazoan GPI-modification motifs consist mainly in variations of preferences to amino acid types at the positions near the omega-site and in the overall motif length. The variations of polypeptide substrates are exploited to suggest a model of the polypeptide binding site of the putative transamidase, the enzyme catalyzing the GPI-modification. The volume of the active site cleft accommodating the four residues omega - 1...omega + 2 appears to be approximately 540 A3.     

Synthesis and pharmacological comparison of dimethylheptyl and pentyl analogs of anandamide

(Dimethylheptyl)anandamide [(16,16-dimethyldocosa-cis-5,8,11,14-tetraenoyl)ethanolamine ] (17a) and its amide analogs were synthesized by Wittig coupling of a ylide derived from a fragment of arachidonic acid. These amides were compared to the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide, 2a) and its amide analogs in pharmacological assays for potential enhancement of cannabimimetic activities. The receptor affinity to rat brain membranes of the dimethylheptyl (DMH) analogs increased by an order of magnitude in most comparisons to the corresponding anandamides in displacement assays versus the cannabinoid agonist [3H]CP 55,940 or antagonist [3H]SR141716A, for which rank order differences in affinity were observed. An order of magnitude enhancement of potency with comparable or higher efficacy in behavioral assays in the mouse tetrad of tests of cannabinoid activity was observed in 17a versus 2a. In contrast, no enhancement in potency for the pentyl to DMH side chain exchange was seen in the mouse vas deferens assay. The data indicate a structural equivalence between classical plant cannabinoids and 2a as well as different receptor-ligand interactions that characterize multiple receptor sites or binding modes.     

Oxytocin modulates glutamatergic synaptic transmission between cultured neonatal spinal cord dorsal horn neurons

The functional characteristics of binding sites for the neuropeptide oxytocin (OT) detected by radioautography in laminae I and II of the dorsal horn (DH) and on cultured neonatal DH neurons were studied on the latter using perforated patch-clamp recordings. The neurons were identified by their spike discharge properties and on the basis of the presence of met-enkephalin-like and glutamate decarboxylase-like immunoreactivities. OT (100 nM) never induced any membrane current at a holding potential of -60 mV but increased the frequency of spontaneously occurring AMPA receptor-mediated EPSCs or the mean amplitude of electrically evoked EPSCs in a subset (35%) of neurons. The frequency of miniature EPSCs (m-EPSCs) recorded in the presence of 0.5 microM tetrodotoxin was also increased by OT (100 nM) without any change in their mean amplitude, indicating an action at a site close to the presynaptic terminal. The decay kinetics of any type of EPSC were never modified by OT. The effect of OT was reproduced by [Thr4, Gly7]-OT (100 nM), a selective OT receptor agonist, and blocked by d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH29]-ornithine vasotocin (100 nM), a specific OT receptor antagonist. Reducing the extracellular Ca2+ concentration from 2.5 to 0.3 mM in the presence of Cd2+ (100 microM) reversibly blocked the effect of OT on m-EPSCs. The OT receptors described here may represent the substrate for modulatory actions of descending hypothalamo-spinal OT-containing pathways on the nociceptive system.