Marked influence of the bridging carbonyl ligands on the photo- and electrochemistry of the clusters [Ru-3(CO)(8)(mu-CO)(2)-(alpha-diimine)] (alpha-diimine = 2,2 '-bipyridine, 4,4 '-dimethyl-2,2 '-bipyridine and 2,2 '-bipyrimidine)

Bonding, photochemical and electrochemical properties of the clusters [Ru-3(CO)(8)(mu-CO)(2)(alpha-diimine)] (alpha-diimine=2,2'-bipyridine (1), 4,4'-dimethyl-2,2'-bipyridine (2) and 2,2'-bipyrimidine (3)) are strongly influenced by the presence of bridging carbonyl ligands. Irradiation at 471 nm initially results in the population of a sigma(Ru-3)pi*(alpha-diimine) excited state. From this state, fast decay takes place to the optically hardly directly accessible pi(Ru/mu-CO) pi*(alpha-diimine) lowest excited state. These assignments agree with theoretical (TD-DFT) results, resonance Raman and picosecond time-resolved infrared spectra. The involvement of the bridging carbonyl ligands in the electron transfer increases the energetic barrier for the formation of open-structure photoproducts such as biradicals and zwitterions. Zwitterions were therefore only obtained in strongly coordinating media such as pyridine at 250 K. The bridging carbonyl ligands also stabilize the radical anions produced upon one-electron reduction of the clusters [Ru-3(CO)(8)(mu-CO)(2)(alpha-diimine)] and observed with cyclic voltammetry, EPR and IR spectroelectrochemistry (for alpha-diimine=2,2'-bipyrimidine). In contrast, open-triangle intermediates formed along the reduction path to [Ru(CO)(2)(alpha-diimine)](n) and [Ru-2(CO)(8)](2-) are more reactive than their triosmium analogues.

Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC.

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.

Night-time tropospheric chemistry of the unsaturated alcohols (Z)-pent-2-en-1-ol and pent-1-en-3-ol: Kinetic studies of reactions of NO3 and N2O5 with stress-induced plant emissions

The night-time tropospheric chemistry of two stress-induced volatile organic compounds (VOCs), (Z)-pent-2-en-1-ol and pent-1-en-3-ol, has been studied at room temperature. Rate coefficients for reactions of the nitrate radical (NO3) with these pentenols were measured using the discharge-flow technique. Because of the relatively low volatility of these compounds, we employed off-axis continuous-wave cavity-enhanced absorption spectroscopy for detection of NO3 in order to be able to work in pseudo first-order conditions with the pentenols in large excess over NO3. The rate coefficients were determined to be (1.53 +/- 0.23) x 10(-13) and (1.39 +/- 0.19) x 10(-14) cm(3) molecule(-1) s(-1) for reactions of NO3 with (Z)-pent-2-en-1-ol and pent-1-en-3-ol. An attempt to study the kinetics of these reactions with a relative-rate technique, using N2O5 as source of NO3 resulted in significantly higher apparent rate coefficients. Performing relative-rate experiments in known excesses of NO2 allowed us to determine the rate coefficients for the N2O5 reactions to be (5.0 +/- 2.8) x 10(-19) cm(3) molecule(-1) s(-1) for (Z)-pent-2-en-1-ol, and (9.1 +/- 5.8) x 10(-19) cm(3) molecule(-1) s(-1) for pent-1-en-3-ol. We show that these relatively slow reactions can indeed interfere with rate determinations in conventional relative-rate experiments.

Regulation of expression of the rat orthologue of mouse double minute 2 (MDM2) by H2O2-induced oxidative stress in neonatal rat cardiac myocytes.

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mm H(2)O(2)) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5' to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H(2)O(2) increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. H(2)O(2) did not detectably increase expression of p53 mRNA or protein but did increase expression of several AP-1 transcription factors. H(2)O(2) increased binding of AP-1 proteins (c-Jun, JunB, JunD, c-Fos, FosB, and Fra-1) to an Mdm2 AP-1 oligodeoxynucleotide probe, and chromatin immunoprecipitation assays showed it increased binding of c-Jun or JunB to the P2 AP-1 RE. Finally, antisense oligonucleotide-mediated reduction of H(2)O(2)-induced Mdm2 expression increased caspase 3 activation. Thus, increased Mdm2 expression is associated with transactivation at the P2 AP-1 RE (rather than the p53 or Ets REs), and Mdm2 induction potentially represents a cardioprotective response to oxidative stress.

Effects of the Allosteric Antagonist 1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea (PSNCBAM-1) on CB1 Receptor Modulation in the Cerebellum

PSNCBAM-1 has recently been described as a cannabinoid CB1 receptor allosteric antagonist associated with hypophagic effects in vivo; however, PSNCBAM-1 effects on CB1 ligand-mediated modulation of neuronal excitability remain unknown. Here, we investigate PSNCBAM-1 actions on CB1 receptor-stimulated [35S]GTPγS binding in cerebellar membranes and on CB1 ligand modulation of presynaptic CB1 receptors at inhibitory interneurone-Purkinje cell (IN-PC) synapses in the cerebellum using whole-cell electrophysiology. PSNCBAM-1 caused non-competitive antagonism in [35S]GTPγS binding studies, with higher potency against the CB receptor agonist CP55940 than for WIN55,212-2 (WIN55). In electrophysiological studies, WIN55 and CP55940 reduced miniature inhibitory postsynaptic currents (mIPSCs) frequency, but not amplitude. PSNCBAM-1 application alone had no effect on mIPSCs; however, PSNCBAM-1 pre-treatment revealed agonist-dependent functional antagonism, abolishing CP55940-induced reductions in mIPSC frequency, but having no clear effect on WIN55 actions. The CB1 antagonist/inverse agonist AM251 increased mIPSC frequency beyond control, this effect was reversed by PSNCBAM-1. PSNCBAM-1 pre-treatment also attenuated AM251 effects. Thus, PSNCBAM-1 reduced CB1 receptor ligand functional efficacy in the cerebellum. The differential effect of PSNCBAM-1 on CP55940 versus WIN55 actions in [35S]GTPγS binding and electrophysiological studies and the attenuation of AM251 effects are consistent with the ligand-dependency associated with allosteric modulation. These data provide the first description of functional PSNCBAM-1 allosteric antagonist effects on neuronal excitability in the mammalian CNS. PSNCBAM-1 allosteric antagonism may provide viable therapeutic alternatives to orthosteric CB1 antagonists/inverse agonists in the treatment of CNS disease.

The decomposition of some RDX and HMX based materials in the one dimensional time to explosion apparatus. Part 2. Estimating the violence of the cook-off event

Various methods of assessment have been applied to the One Dimensional Time to Explosion (ODTX) apparatus and experiments with the aim of allowing an estimate of the comparative violence of the explosion event to be made. Non-mechanical methods used were a simple visual inspection, measuring the increase in the void volume of the anvils following an explosion and measuring the velocity of the sound produced by the explosion over 1 metre. Mechanical methods used included monitoring piezo-electric devices inserted in the frame of the machine and measuring the rotational velocity of a rotating bar placed on the top of the anvils after it had been displaced by the shock wave. This last method, which resembles original Hopkinson Bar experiments, seemed the easiest to apply and analyse, giving relative rankings of violence and the possibility of the calculation of a “detonation” pressure.

Structural characterization of a new crystal form of the four-way Holliday junction formed by the DNA sequence d(CCGGTACCGG)2: sequence versus lattice?

DNA-strand exchange is a vital step in the recombination process, of which a key intermediate is the four-way DNA Holliday junction formed transiently in most living organisms. Here, the single-crystal structure at a resolution of 2.35 Å of such a DNA junction formed by d(CCGGTACCGG)2, which has crystallized in a more highly symmetrical packing mode to that previously observed for the same sequence, is presented. In this case, the structure is isomorphous to the mismatch sequence d(CCGGGACCGG)2, which reveals the roles of both lattice and DNA sequence in determining the junction geometry. The helices cross at the larger angle of 43.0° (the previously observed angle for this sequence was 41.4°) as a right-handed X. No metal cations were observed; the crystals were grown in the presence of only group I counter-cations.

Steric stabilisation of the P_P bond in a bulky tetraorganodiphosphine: synthesis, characterisation and X-ray structure determination of tetrakis(2,4,6-triisopropylphenyl)diphosphine

We report the synthesis and characterisation of tetrakis(2,4,6-triisopropylphenyl)diphosphine. Synthesis is effected by the treatment of PCl3 with an excess of 2,4,6-triisopropylphenyllithium (or the equivalent Grignard reagent) in 70% yield. While under normal circumstances the triarylphosphine would be expected, excessive bulk prevents this, and the resulting diphosphine is, unusually, stable to PP cleavage by further organolithium moieties. The compound is stable, both thermally (m.p. 185°C) and to air and water in the solid state, although conversion to the equivalent diorganophosphinate ester is effected by boiling ethanolic solutions in air. Crystallisation from hexane/ethanol afforded pale yellow crystals of X-ray quality. The molecule is characterised by m.p., IR, NMR, elemental analysis (C, H, P) and MS. The X-ray structure shows an antiperiplanar conformation with a PP separation of 2.2461(16) Å. Comparisons are made with other diphosphines, the title compound being only the fourth simple diphosphine to be structurally characterised.

Determination by MAD-DM of the structure of the DNA duplex d[ACGTACG(5-BrU)]2 at 1.46 Å and 100 K

A four-wavelength MAD experiment on a new brominated octanucleotide is reported here. d[ACGTACG(5-BrU)], C77H81BrN30O32P7, (DNA) = 2235, tetragonal, P43212 (No. 96), a = 43.597, c = 26.268 Å, V = 49927.5 Å3, Z = 8, T = 100 K, R = 10.91% for 4312 reflections between 15.0 and 1.46 Å resolution. The self-complementary brominated octanucleotide d[ACGTACG(5-BrU)]2 has been crystallized and data measured to 1.45 Å at both 293 K and a second crystal flash frozen at 100 K. The latter data collection was carried out to the same resolution at the four wavelengths 0.9344, 0.9216, 0.9208 and 0.9003 Å, around the Br K edge at 0.92 Å and the structure determined from a map derived from a MAD data analysis using pseudo-MIR methodology, as implemented in the program MLPHARE. This is one of the first successful MAD phasing experiments carried out at Sincrotrone Elettra in Trieste, Italy. The structure was refined using the data measured at 0.9003 Å, anisotropic temperature factors and the restrained least-squares refinement implemented in the program SHELX96, and the helical parameters are compared with those previously determined for the isomorphous d(ACGTACGT)2 analogue. The asymmetric unit consists of a single strand of octamer with 96 water molecules. No countercations were located. The A-DNA helix geometry obtained has been analysed using the CURVES program.

The anions [(ZrOH(CO3)3)2]6− and [(ZrOH(C2O4)3)2]6−: single crystal X-ray diffraction studies of the complexes guanidinium zirconium carbonate [(C(NH2)3)3ZrOH(CO3)3·H2O]2 and sodium zirconium oxalate Na6(ZrOH(C2O4)3)2·7H2O

The complex [(C(NH2)3)3ZrOH(CO3)3·H2O]2 (A) has been shown by means of a single crystal X-ray diffraction study to contain [C(NH2)3]+ cations and dimeric anions of formulation [(ZrOH(CO3)3)2]6−. The anion is centrosymmetric with each metal being bonded to two bridging OH groups and three chelating CO2−3 ions. The Zr atoms are thus eight coordinate with a dodecahedral environments. The ZrO distances formed by the bridgng OH groups are shorter than those formed through zirconiu carbonate interactions. The non-bonded Zr…Zr distance is 3.47(2) Å. An infrared spectroscopic investigation of A provides data which support the findings of the crystallographic study. Likewise the complex Na6(ZrOH(CO2O4)3)2·7H2O (B) contains the anion [(ZrOH(C2O4)3)2]6−. This anion is structurally related to the anion in A as each Zr atom has an eight-coordinate dodecahedral environment being bonded to two bridging OH groups and three chelating oxalate ligands, but has no imposed crysallographic symmetry. The Zr…Zr non-bonded distance is 3.50(1) Å. The OZrO bridge angles are 69.7(4)° and A and 67.4(3)° in B.

The synthesis of dicobalt and dinickel complexes of trimethylsilylethynyl-1, 2-dihydrofullerene; characterisation by n.m.r. and structure of the first acyclic metal fullerene derivative: molecular structure of [η2-{2-H-1-(Me3SiC ≡ C)-C60}Ni2(η-C5H5)2]

The synthesis and characterisation of the complexes [η2-{2-H-1-(Me3SiC ≡ C)-C60}Co2(CO)6] (2)} and [η-2-{2-H-1-(Me3SiC ≡ C)-C60}Ni2η-C5H5)2] (3)} is reported, together with a single-crystal molecular structure for (3). This provides the first structural data for an acyclic metal derivative of [60]-fullerene.

Structure of the DNA octanucleotide d(ACGTACGT)2

d(ACGTACGT), C78H84N30O32P7.20H2O, Mr (DNA) = 2170, tetragonal, P43212 (No 96), a = 42.845 (1), b = 42.845(1), c = 24.804 (1) Å, V = 45532.5 (2) Å3, z = 8,(MoK) = 0.71069 Å,µ(MoK) = 0.10 mm-1, T = 295 K, R = 0.18 for 1994 unique reflections between 5.0 and 1.9 Å resolution. The self-complementary octanucleotide d(ACGTACGT)2 has been crystallized and its structure determined to a resolution of 1.9 Å. The asymmetric unit consists of a single strand of octamer with 20 water molecules. It is only the second example of an octanucleotide having terminal A·T base pairs whose structure has been determined by X-ray crystallography. The sequence adopts the modified A-type conformation found for all octanucleotide duplexes studied to date with the helix bent by approximately 15° and an average tilt angle of 0°. Unusually the data collection was carried out using a 3 kW molybdenum sealed-tube source. The conformational details are discussed in comparison with other closely related sequences.

Synthesis, X-ray crystal structure and catalytic activity of the manganese(II) complex [Mn(bdoa)(H2O)3](bdoaH2 =benzene-1,2-dioxyacetic acid)

Benzene-1,2-dioxyacetic acid (bdoaH2) reacts with Mn(CH3CO2)2·4H2O in an ethanol-water mixture to give the manganese(II) complex [Mn(bdoa)(H2O)3]. The X-ray crystal structure of the complex shows the metal to be pseudo seven-coordinate. The quadridentate bdoa2− dicar☐ylate ligand forms an essentially planar girdle around the metal, being strongly bondedtransoid by a car☐ylate oxygen atom from each of the two car☐ylate moieties (mean MnO 2.199A˚) and also weakly chelated by the two internal ether oxygen atoms (mean MnO 2.413A˚). The coordination sphere about the manganese is completed by three water molecules (mean MnO 2.146A˚) lying in a meridional plane orthogonal to that of the bdoa2− ligand. Magnetic, conductivity and voltammetry data for the complex are given, and its use as a catalyst for the disproportionisation of H2O2 is described.