Morphine-3-glucuronide's neuro-excitatory effects are mediated via indirect activation of N-methyl-D-aspartic acid receptors: Mechanistic studies in embryonic cultured hippocampal neurones

Indirect evidence indicates that morphine-3-glucuronide (M3G) may contribute significantly to the neuro-excitatory side effects (myoclonus and allodynia) of large-dose systemic morphine. To gain insight into the mechanism underlying M3G' s excitatory behaviors, We used fluo-3 fluorescence digital imaging techniques to assess the acute effects of M3G (5-500 muM) on the cytosolic calcium concentration ([Ca2+](CYT)) in cultured embryonic hippocampal neurones. Acute (3 min) exposure of neurones to M3G evoked [Ca2+](CYT) transients that were typically either (a) transient oscillatory responses characterized by a rapid increase in [Ca2+](CYT) oscillation amplitude that was sustained for at least similar to30 s or (b) a sustained increase in [Ca2+](CYT) that slowly recovered to baseline. Naloxone-pretreatment decreased the proportion of M3G-responsive neurones by 10%-25%, implicating a predominantly non-opioidergic mechanism. Although the naloxone-insensitive M3G-induced increases in [Ca2+](CYT) were completely blocked by N-methyl-D-aspartic acid (NMDA) antagonists and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (alphaamino-3-hydroxy-5-methyl-4-isoxazolepropiordc acid/ kainate antagonist), CNQX did not block the large increase in [Ca2+](CYT) evoked by NMDA (as expected), confirming that N13G indirectly activates the NMDA receptor. Additionally, tetrodotoxin (Na+ channel blocker), baclofen (gamma-aminobutyric acid, agonist), MVIIC (P/Q-type calcium channel blocker), and nifedipine (L-type calcium channel blocker) all abolished M3G-induced increases in [Ca2+](CYT), suggesting that M3G may produce its neuro-excitatory effects by modulating neurotransmitter release. However, additional characterization is required.

Differential expression of N-methyl-D-aspartate receptor NR2 isoforms in Alzheimer's disease

We have previously shown that the expression of NMDA receptor NR1 subunit mRNA splice variants in Alzheimer's disease (AD) brain varies according to regional susceptibility to pathological damage. Here we investigated the expression of the modulatory NR2 subunits of the NMDA receptor using quantitative RT-PCR to assay all NR2 isoforms. Significantly lower expression of NR2A and NR2B transcripts was found in susceptible regions of AD brain, whereas expression of NR2C and NR2D transcripts did not differ from that in controls. Western blot analysis confirmed a lower expression of the NR2A and NR2B isoforms at the protein level. The results suggest that NR2 subunit composition may modulate NMDA receptor-mediated excitotoxicity. NMDA receptor dysfunction might give rise to the regionally selective pattern of neuronal loss that is characteristic of AD.

The preparation and characterization of tin(IV) complexes of 2-quinolinecarboxaldehyde Schiff bases of S-methyl- and S-benzyldithiocarbazates and the X-ray crystal and molecular structures of the 2-quinolinecarboxaldehyde Schiff base of S-benzyldithi

New tin(IV) complexes of empirical formula, Sn(NNS)I-3 (NNS = anionic forms of the 2-quinolinecarboxaldehyde Schiff bases of S-methyl- and S-benzyldithiocarbazate) have been prepared and characterized by a variety of physico-chemical techniques. In the solid state, the Schiff bases exist as the thione tautomer but in solution and in the presence of tin(IV) iodide they convert to the thiol tautomer and coordinate to the tin atom in their deprotonated thiolate forms. The structures of the free ligand, Hqaldsbz and its triiodotin(IV) complex, [Sn(qaldsbz)I-3] have been determined by X-ray diffraction. The complex, [Sn(qaldsbz)I-3] has a distorted octahedral structure with the Schiff base coordinated to the tin atom as a uninegatively charged tridentate chelating agent via the quinoline nitrogen atom, the azomethine nitrogen atom and the thiolate sulfur atom. The three iodo ligands are coordinated meridionally to the tin atom. The distortion from an ideal octahedral geometry of [Sn(qaldsbz)I-3] is attributed to the restricted bite size of the tridentate Schiff base ligand. (C) 2004 Elsevier Ltd. All rights reserved.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Accessing chain length dependent termination rate coefficients of methyl methacrylate (MMA) via the reversible addition fragmentation chain transfer (RAFT) process

The RAFT-CLD-T methodology is demonstrated to be not only applicable to 1-substituted monomers such as styrene and acrylates, but also to 1,1-disubstituted monomers such as MMA. The chain length of the terminating macromolecules is controlled by CPDB in MMA bulk free radical polymerization at 80 degrees C. The evolution of the chain length dependent termination rate coefficient, k(t)(i,i), was constructed in a step-wise fashion, since the MMA/CPDB system displays hybrid behavior (between conventional and living free radical polymerization) resulting in initial high molecular weight polymers formed at low RAFT agent concentrations. The obtained CLD of k(t) in MMA polymerizations is compatible with the composite model for chain length dependent termination. For the initial chain-length regime, up to a degree of polymerization of 100, k(t) decreases with alpha (in the expression k(t)(i,i) = k(t)(0) . i(-alpha)) being close to 0.65 at 80 degrees C. At chain lengths exceeding 100, the decrease is less pronounced (affording an alpha of 0.15 at 80 degrees C). However, the data are best represented by a continuously decreasing nonlinear functionality implying a chain length dependent alpha.

Controlled Radical Polymerization of Styrene and Methyl Acrylate in the Presence of Reversible Addition-Fragmentation Chain Transfer Agents, Phenylethyl Phenyl Dithioacetate and Phenyldithioacetic Acid

The use of phenyldithioacetic acid (PDA) in homopolymerizations of styrene or methyl acrylate produced only a small fraction of chains with dithioester end groups. The polymerizations using 1-phenylentyl phenyldithioacetate (PEPDTA) and PDA in the same reaction showed that PDA had little or no influence on the rate or molecular weight distribution even when a 1:1 ratio is used. The mechanistic pathway for the polymerizations in the presence of PDA seemed to be different for each monomer. Styrene favors addition of styrene to PDA via a Markovnikov type addition to form a reactive RAFT agent. The polymer was shown by double detection SEC to contain dithioester end groups over the whole distribution. This polymer was then used in a chain extension experiment and the M-n was close to theory. A unique feature of this work was that PDA could be used to form a RAFT agent in situ by heating a mixture of styrene and PDA for 24 h at 70 degrees C and then polymerizing in the presence of AIBN to give a linear increase in Mn and low values of PDI (< 1.14). In the case of the polymerization of MA with PDA, the mechanism was proposed to be via degradative chain transfer. (c) 2005 Wiley Periodicals, Inc.

Diphenyltin(IV) complexes of the 2-quinolinecarboxaldehyde Schiff bases of S-methyl- and S-benzyldithiocarbazate (Hqaldsme and Hqaldsbz): X-ray crystal structures of Hqaldsme and two conformers of its diphenyltin(IV) complex

New organometallic tin(IV) complexes of the empirical formula Sn(NNS)Ph2Cl (NNS = anionic forms of the 2-quinolinecarboxaldehyde Schiff bases of S-methyl- and S-benzyldithiocarbazate) have been prepared and characterized by IR, electronic, I H NMR and ES mass spectroscopic techniques. The molecular structures of the 2-quinolinecarboxaldehyde Schiff base of S-methyldithiocarbazate (Hqaldsme) and its diphenyltin(IV) complex, Sn(qaldsme)Ph2Cl, have been determined by X-ray diffraction. In the solid state, the ligand remains as the thione tautomer in which the dithiocarbazate chain adopts an E,E configuration and is almost coplanar with the quinoline ring. The Sn(qaldsme)Ph2Cl complex crystallizes in two distinctly different conformationally isomeric forms, each having the same space group but different lattice parameters. X-ray analysis shows that in each polymorph, the tin atom adopts a distorted octahedral geometry with the Schiff base coordinated to it as a uninegatively charged tridentate chelating agent via the quinoline nitrogen atom, the azomethine nitrogen atom and the thiolate sulfur atom. The two phenyl groups occupy axial positions and the chloride ligand occupies the sixth coordination position of the tin atom. The deprotonated ligand adopts an E,E,Z configuration in the complex. (C) 2004 Elsevier Ltd. All rights reserved.

Transcriptional response of sugarcane roots to methyl jasmonate

The effect of methyl jasmonate treatment on gene expression in sugarcane roots signalling between roots and shoots was studied. A collection of 829 ESTs were obtained from sugarcane roots treated with the defence-regulator methyl jasmonate (MJ) treatment. A subset of 747 of these were combined with 4793 sugarcane ESTs obtained from stem tissues in a cDNA microarray and experiments undertaken to identify genes that were induced in roots 24-120 h following treatment with MJ. Two data analysis systems (t-statistic and tRMA) were used to analyse the microarray results and these methods identified a common set of 21 ESTs corresponding to transcripts significantly induced by MJ in roots and 23 that were reduced in expression following MJ treatment. The induction of six transcripts identified in the microarray analysis was tested and confirmed using northern blotting. Homologues of genes encoding lipoxygenase and PR-10 proteins were induced 824 It after MJ treatment while the other four selected transcripts were induced at later time points. Following treatment of roots with MJ, the lipoxygenase homologue, but not the PR-10 homologue, was induced in untreated stem and leaf tissues. The PR-10 homologue and a PR-1 homologue, but not the lipoxygenase homologue, were induced in untreated tissues after the application of SA to roots. Repeated foliar application of MJ had no apparent effects on plant growth and was demonstrated to increase lipoxygenase transcripts in roots, but did not increase transcript levels-of other genes tested. These results lay a foundation for further studies of induced pest and disease resistance in sugarcane roots. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

The effects of alanine-substituted conantokin-G and ifenprodil on the human spermine-activated N-methyl-D-aspartate receptor

We evaluated the effects of Ala-7-conantokin-G (Con-G(A7)) and ifenprodil on the modulation by spermine of [H-3]MK801 binding to human cortical membranes. Human cortical tissue was obtained at autopsy and stored at -80 degreesC until assay. Both Con-GA7 and ifenprodil inhibited [H-3]MK801 binding, but spermine affected these inhibitions differently. Con-G(A7) IC50 changed little with spermine concentration, indicative of a non-competitive interaction, whereas the rightward shift in ifenprodil IC50 with increasing spermine concentration suggested partial competition. When the two agents were tested against the biphasic activation of [H-3]MK801 binding by spermine, they again differed in their effects. In the activation phase Con-G(A7) was a non-competitive inhibitor of spermine activation, and may even enhance the spermine EC50, while the ifenprodil data indicated a partially competitive interaction. Both agents were non-competitive in the inhibitory phase. Overall, the data suggest that Con-G(A7) and ifenprodil interact differently with the polyamine modulation of the glutamate-N-methyl-D-aspartate receptor. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

Methyl jasmonate vapour treatment suppresses specking caused by Botrytis cinerea on cut Freesia hybrida L. flowers

Treatment of cut freesia var. Cote d'Azur flowers with methyl jasmonate (MeJA, 0.1 mu l MeJA l(-1)) vapour suppressed petal specking caused by Botrytis cinerea infection. MeJA efficacy was concentration and incubation temperature dependent. Disease severity, lesion numbers and lesion diameters decreased with increasing MeJA concentration from 0.025 to 0.1 mu l MeJA l(-1). However, there were no significant (P > 0.05) differences among MeJA concentrations examined. MeJA was more effective in reducing B. cinerea flower specking at 20 degrees C than at 12 degrees C. MeJA treatment was ineffective at 5 degrees C. At 20 degrees C, MeJA treatment at 0.1 mu l MeJA l(-1) reduced disease severity, lesion numbers and lesion diameters by 58, 50 and 48%, respectively, as compared to untreated controls. In a repeat experiment, disease severity, lesion numbers and lesion diameters on MeJA vapour treated flowers after 12 h of incubation were reduced by 68, 56 and 50%, respectively. MeJA did not exert direct antifungal activity in-vitro, suggesting that treatment in-vivo reduced B. cinerea-induced flower specking by induction of host defence responses. MeJA at 0.1 mu l MeJA l(-1) significantly (P < 0.05) increased vase life of cut freesia flowers and delayed senescence judged by lower wilt scores and higher fresh weights as compared to untreated controls. (c) 2005 Elsevier B.V. All rights reserved.

Organic-inorganic poly(methyl methacrylate) hybrids with confined polyhedral oligosilsesquioxane macromonomers

We herein report the synthesis of organic-inorganic hybrid poly(methyl methacrylate) containing 1 polyhedral oligosilsesquioxanes. Octakis(3-hydroxypropyldimethylsiloxy)octasilsesquioxane (OHPS) was synthesized from octakis(hydridodimethylsiloxy)octasilsesquioxane [Si8O12(OSiMe2H)(8), Q(8)M(8)(H)] following literature procedures. Octakis(tnethacryloxypropyldimethylsiloxy) octasilsesquioxane (OMPS) was synthesized via the reaction of methacryloyl chloride or methacrylic acid anhydride with OHPS, with the latter giving improved purity. Polymerization of OMPS with methyl inethacrylate using a dibenzoylperoxide initiator gave a highly cross-linked polymer. Characterization of the polymer was performed using Fourier transform IR spectroscopy, Si-29 NMR, differential scanning calorimetry, thermogravimetric analysis, atomic force microscopy, and transmission electron microscopy with energy-dispersive X-ray analysis. The polymer was found to be largely homogeneous. Increasing the OMPS concentration in the polymer gave increased decomposition and glass transition temperatures.

Synthetic, EPR spectroscopic, magnetic and X-ray crystallographic structural studies on copper(II) complexes of the tridentate N2S donor ligand formed from 6-methyl-2-formylpyridine and S-methyldithiocarbazate (Hmpsme)

New copper(II) complexes of general empirical formula, Cu(mpsme)X center dot xCH(3)COCH(3) (mpsme = anionic form of the 6-methyl-2-formylpyridine Schiff base of S-methyldithiocarbazate; X = Cl, N-3, NCS, NO3; x = 0, 0.5) have been synthesized and characterized by IR, electronic, EPR and susceptibility measurements. Room temperature mu(eff) values for the complexes are in the range 1.75-2.1 mu(beta) typical of uncoupled or weakly coupled Cu(II) centres. The EPR spectra of the [Cu(mpsme)X] (X = Cl, N-3, NO3, NCS) complexes reveal a tetragonally distorted coordination sphere around the mononuclear Cu(II) centre. We have exploited second derivative EPR spectra in conjunction with Fourier filtering (sine bell and Hamming functions) to extract all of the nitrogen hyperfine coupling matrices. While the X-ray crystallography of [Cu(mpsme)NCS] reveals a linear polymer in which the thiocyanate anion bridges the two copper(II) ions, the EPR spectra in solution are typical of a magnetically isolated monomeric Cu(II) centres indicating dissociation of the polymeric chain in solution. The structures of the free ligand, Hmpsme and the {[Cu(mpsme)NO3] center dot 0.5CH(3)COCH(3)}(2) and [Cu(mpsme)NCS](n) complexes have been determined by X-ray diffraction. The {[Cu(mpsme)NO3]0.5CH(3)COCH(3)}(2) complex is a centrosymmetric dimer in which each copper atom adopts a five-coordinate distorted square-pyramidal geometry with an N2OS2 coordination environment, the Schiff base coordinating as a uninegatively charged tridentate ligand chelating through the pyridine and azomethine nitrogen atoms and the thiolate, an oxygen atom of a unidentate nitrato ligand and a bridging sulfur atom from the second ligand completing the coordination sphere. The [Cu(mpsme)(NCS)](n) complex has a novel staircase-like one dimensional polymeric structure in which the NCS- ligands bridge two adjacent copper(II) ions asymmetrically in an end-to-end fashion providing its nitrogen atom to one copper and the sulfur atom to the other. (c) 2005 Elsevier B.V. All rights reserved.

Effects of postharvest methyl jasmonate treatments against Botrytis cinerea on Geraldton waxflower (Chamelaucium uncinatum)

Cut Geraldton waxflower (Chamelaucium uncinatum Schauer) flowers are often infected with Botrytis cinerea. Release of infection from quiescence can cause ethylene production by invaded host tissues and result in flower abscission. Postharvest floral organ abscission is a major problem for the commercial waxflower industry. Methyl jasmonate (MeJA) occurs naturally in plant tissue and has a signalling role in eliciting induced systemic resistance against disease. MeJA treatments have been shown to suppress B. cinerea infecting cut rose flowers. The present experiments investigated the potential of exogenous MeJA treatments for B. cinerea management on harvested waxflower. MeJA treatments of 10 and 100 L liquid MeJA/L of air applied to cv. Purple Pride and 1 L MeJA/L to cv. Mullering Brook gave reductions in disease severity for uninoculated stems. However, concentrations of 100 L MeJA/L applied to Purple Pride in addition to 1 and 10 L MeJA/L applied to Mullering Brook increased the incidence of floral organ fall. Flower abscission upon treatment with MeJA may be due to induced systemic resistance-associated upregulation of ethylene biosynthesis. MeJA treatments had no direct effect on B. cinerea hyphal elongation in vitro. Collectively, these results show that while MeJA treatment may elicit defence in waxflower against Botrytis, the chemical also causes floral organ fall. Thus, exogenous MeJA treatments do not have potential for B. cinerea management on harvested waxflower.

Acibenzolar-S-methyl and methyl jasmonate treatments of glasshouse-grown freesias suppress post-harvest petal specking caused by Botrytis cinerea

Compounds that activate host plant defence responses potentially offer socio-environmentally sound alternative methods for disease control. In a series of glasshouse trials over 2 years, pre-harvest sprays with acibenzolar-S-methyl (ASM) and methyl jasmonate (MeJA) were tested for suppression of post-harvest infection of cut Freesia hybrida L. flowers by Botrytis cinerea. For the ASM treatments, variability in reducing the incidence of B. cinerea disease was observed between years freesia varieties, incubation temperatures and ASM concentrations. In the first year, the greatest reductions in lesion numbers on ASM-treated var. 'Cote d'Azur' were recorded using 2.86 mM ASM. For three different post-harvest temperature regimes, the relative reductions in lesion numbers, compared to untreated controls, were 45% at 5 degrees C, 40% at 12 degrees C and 30% at 20 degrees C, respectively. In the second year, lesion numbers were most reduced using 1.43 mM ASM to treat freesia var. 'Dukaat' flowers. Here, the relative reductions were to 44% at 5 degrees C, 26% at 12 degrees C and 51% at 20 degrees C. MeJA treatments were, in general, more consistently effective than ASM treatments in reducing lesion numbers and lesion diameters on cut freesia flowers. MeJA-treated (0.2 mM) freesia flowers (var. 'Dukaat') incubated at 20 degrees C showed relative reductions of 62%, and 45% for lesion number and lesion diameter, respectively. The differing efficacy between ASM and MeJA treatments could be attributed to their differential abilities to induce the salicylic acid (SA)-mediated vs. the jasmonic acid (JA)-mediated host defence pathways, respectively.

Simulation of glassy state relaxations in polymers: A static analysis of methyl group and methoxy group rotation in poly(vinyl methyl ether)

We show that the simple quasi-static technique, also called the adiabatic mapping technique, can be used to determine the energetics of rotation of methyl and methoxy groups in amorphous poly(vinyl methyl ether) even though the latter process is too slow to be amenable to direct molecular dynamics simulation. For the methyl group rotation, we find that the mean and standard deviation of the simulated rotational barrier heights agree well with experimental data from quasi-elastic neutron scattering. In the case of the methoxy groups we find that just 4% of the groups contribute more than 90% of the observed dielectric relaxation strength. The groups which make the most contribution are those which, by virtue of their particular conformation and local environment, have two alternative positions of similar energy.