The crystal structure of a complex of Campylobacter jejuni dUTPase with substrate analogue sheds light on the mechanism and suggests the "basic module" for dimeric d(C/U)TPases

The crystal structure of the dUTPase from the important gastric pathogen Campylobacter jejuni has been solved at 1.65 A spacing. This essential bacterial enzyme is the second representative of the new family of dimeric dUTPases to be structurally characterised. Members of this family have a novel all-alpha fold and are unrelated to the all-beta dUTPases of the majority of organisms including eukaryotes such as humans, bacteria such as Escherichia coli, archaea like Methanococcus jannaschii and animal viruses. Therefore, dimeric dUTPases can be considered as candidate drug targets. The X-ray structure of the C.jejuni dUTPase in complex with the non-hydrolysable substrate analogue dUpNHp allows us to define the positions of three catalytically significant phosphate-binding magnesium ions and provides a starting point for a detailed understanding of the mechanism of dUTP/dUDP hydrolysis by dimeric dUTPases. Indeed, a water molecule present in the structure is ideally situated to act as the attacking nucleophile during hydrolysis. A comparison of the dUTPases from C.jejuni and Trypanosoma cruzi reveals a common fold with certain distinct features, both in the rigid and mobile domains as defined in the T.cruzi structure. Homologues of the C.jejuni dUTPase have been identified in several other bacteria and bacteriophages, including the dCTPase of phage T4. Sequence comparisons of these proteins define a new superfamily of d(C/U)TPases that includes three distinct enzyme families: (1) dUTPases in trypanosomatides, C.jejuni and several other Gram-negative bacteria, (2) predicted dUTPases in various Gram-positive bacteria and their phages, and (3) dCTP/dUTPases in enterobacterial T4-like phages. All these enzymes share a basic module that consists of two alpha-helices from the rigid domain, two helices from the mobile domain and connecting loops. These results in concert with a number of conserved residues responsible for interdomain cross-talk provide valuable insight towards rational drug design.     

Synthesis and structural characterisation of new cationic dinuclear ruthenium(II) thiolato complexes of the type [Ru2(η-arene)2(μ-p-S-C6H4-Br)3]

Dinuclear dichloro complexes [Ru(C₆H₆)Cl₂]₂, [Ru(p-MeC₆H₄ ⁱPr)Cl₂]₂, [Ru(1,2,4,5-C₆H₂Me₄)Cl₂]₂, and [Ru(C₆Me₆)Cl₂]₂ react in ethanol with p-bromothiophenol to give the corresponding cationic complexes [Ru₂(C₆H₆)₂(p-S-C₆H₄-Br)₃]⁺ (1), [Ru₂(p-MeC₆H₄ ⁱPr)₂(p-S-C₆H₄-Br)₃]⁺ (2), [Ru₂(1,2,4,5-C₆H₂Me₄)₂(p-S-C₆H₄-Br)₃]⁺ (3), and [Ru₂(C₆Me₆)₂(p-S-C₆H₄-Br)₃]⁺ (4), which can be isolated in quantitative yield as their chloride salts. X-ray structure analysis of these complexes shows that the nature of the arene ligand influences the folding of the p-S-C₆H₄-Br units. In 1, where the less hindered arene ligand is present, the three phenyl rings of the thiolato units are not constrained to a coplanar arrangement, whereas in 4 the C₆Me₆ forces the three phenyl rings to be in perfect planarity. Complexes 2 and 3 show an intermediary arrangement.     

Structural, spectroscopic and redox studies of a new ruthenium(III) complex with an imidazole-rich tripodal ligand

The present paper describes a new tripodal ligand containing imidazole and pyridine arms and its first cis-[Ru^III(L)(Cl)₂]ClO₄ complex (1). The crystal structure of 1 shows Ru^III in a distorted octahedral geometry, in which two chloride ions, cis-positioned to each other, are coordinated besides the four nitrogen atoms from the tetradentate ligand L. The cyclic voltammogram of 1 exhibits three redox processes at −67, +73 and +200 mV versus SCE, which are attributed to the Ru^III/Ru^II couple in the cis-[Ru^III(L)(Cl)₂]⁺, cis-[Ru^II(L)(H₂O)(Cl)]⁺ and cis-[Ru^II(L)(H₂O)₂]²⁺, respectively. After chemical reduction (Zn(Hg) or Eu^II) only the cis-[Ru^II(L)(H₂O)₂]²⁺ species is observed in the cyclic voltammetry. Complex 1 absorbs at 470 nm (ε=1.4×10³ mol⁻¹ L cm⁻¹), 335 nm (ε=7.9×10³ mol⁻¹ L cm⁻¹), 301 nm (ε=6.7×10³ mol⁻¹ L cm⁻¹) and 264 nm (ε=9.9×10³ mol⁻¹ L cm⁻¹), in water solution (CF₃COOH, 0.01 mol L⁻¹, μ=0.1 mol L⁻¹ with CF₃COONa). Spectroelectrochemical experiments show a decrease of the bands at 335 and 301 nm, which are attributed to LMCT transitions from the chloride to the Ru^III center and the appearance of a broad band at 402 nm ascribed to MLCT transition from the Ru^II center to the pyridine ligand. The lability of the water ligands in the cis-[Ru^II(L)(H₂O)₂]²⁺ species has been investigated using the auxiliary ligand pyrazine. Reactions in the presence of stoichiometric and excess of pyrazine yield the same species, cis-[Ru^II(L)(H₂O)(pz)]²⁺, which exhibits a reversible redox process at 493 mV versus SCE and absorbs at 438 nm (ε=5.1×10³ mol⁻¹ L cm⁻¹) and 394 nm (ε=4.2×10³ mol⁻¹ L cm⁻¹). Experiments performed with a large excess of pyrazine gave a specific rate constant k₁=(2.8±0.5)×10⁻² M⁻¹ s⁻¹, at 25 °C, in CF₃COOH, 0.01 mol L⁻¹, μ=0.1 mol L⁻¹ (with CF₃COONa).     

New dinuclear arene ruthenium complexes with P,S- or P,O-chelating ligands

Two equivalents of 2-diphenylphosphinobenzoic acid react with 1,2-ethanedithiol and 1,8-diaminonaphthalene under peptidic coupling conditions to give the new ligands 1,2-bis-S-[2^′-(diphenylphosphino)benzoyl]dithioethane (dppte) (1) and 1,2-bis-N-[2^′-(diphenylphosphino)benzoyl]diaminonaphthalene (dppan) (2), respectively. 1 and 2 have been characterised by mass spectrometry, elemental analysis, NMR, IR spectroscopy, and by single-crystal X-ray structure analysis. 2 is easily oxidised by air to give the monophosphine oxide derivatives (3). Single-crystal X-ray structure analysis of 3 shows an intramolecular hydrogen bond between an amido and the phosphoryl oxygen atom. Compounds 1 and 2 react with [RuCl₂(η⁶-p-cymene)]₂ to give the dinuclear complexes [RuCl(η⁶-p-cymene)(dppte)RuCl(η⁶-p-cymene)]²⁺ (4) and [RuCl(η⁶-p-cymene)(dppan)RuCl(η⁶-p-cymene)]²⁺ (5). As determined by single-crystal X-ray structure analysis, 4 and 5 adopt different coordination modes to the ruthenium atoms. In 4 the symmetric dppte ligand is P,S coordinated to the ruthenium atom, whereas in 5 the dppan ligand prefers a P,O coordination mode.     

Synthesis, characterization and structural diversity of lanthanide chlorides supported by the β-diketiminate ligand [{(2,6-Me2C6H3)NC(Me)}2CH]

Reactions of the β-diketiminate lithium salt L²Li [L²={(2,6-Me₂C₆H₃)NC(Me)}₂CH] with anhydrous LnCl₃ (Ln=Yb, Sm, Nd) in 1:1 molar ratio in THF afforded the new β-diketiminate lanthanide complexes L²LnCl(THF)(μ-Cl)₂Li(THF)₂ (Ln=Yb (1), Sm (2), Nd (3)). Recrystallization of complexes 1-3 from toluene gave the neutral complexes L²LnCl₂(THF)₂ (Ln=Yb (4), Sm (5), Nd (6)). Recrystallization of complexes 4 and 5 in hot toluene for two times gave the dinuclear complexes L²ClLn(μ-Cl)₃LnL²(THF) (Ln=Yb (7), Sm (8)). Treatment of the mother liquor of complex 2 in hot toluene for three times gave the novel trinuclear complex L²SmCl(μ-Cl)₃SmL²(μ-Cl)Li(L²H)(THF) (9). Each of these complexes was well characterized, while complexes 3, 7 and 9 have been characterized by X-ray diffraction structure determination.     

Synthesis and structural characterization of trans-[Mo2(H2DMepyF)2(CH3CN)4](BF4)6 (HDMepyF=N,N-di(6-methyl-2-pyridyl)formamidine)

Reaction of Mo₂(O₂CCH₃)₂(DMepyF)₂ (HDMepyF=N,N^′-di(6-methyl-2-pyridyl)formamidine) with HBF₄ in CH₂Cl₂/CH₃CN afforded the complex trans-[Mo₂(H₂DMepyF)₂(CH₃CN)₄](BF₄)₆ (1), which crystallized in two forms, trans-[Mo₂(H₂DMepyF)₂(CH₃CN)₄](ax-CH₃CN)₂(BF ₄)₆ · 2CH₃CN (1a), and trans- [Mo₂(H₂DMepyF)₂(CH₃CN)₄](ax-BF₄) ₂(BF₄)₄ · 2CH₃CN (1b). The molecular structures of complexes (1) consist of two quadruply bonded molybdenum atoms, which are spanned by two trans-bridging formamidinate ligands and coordinated by four trans-CH₃CN. Each H₂DMepyF⁺ ligand adopts an s-cis,s-cis- conformation. The difference between 1a and 1b is that complex 1a contains two CH₃CN molecules as axial ligands, while 1b contains two BF₄⁻ anions as axial ligands. Complex 1 is the first dimolybdenum complex containing a pair of trans bridging ligands and two pairs of trans-CH₃CN ligands.     

A high-capacity assay for PPARgamma ligand regulation of endogenous aP2 expression in 3T3-L1 cells

A novel class of insulin-sensitizing agents, the thiazolidinedines (TZDs), has proven effective in the treatment of type 2 diabetes. These compounds, as well as a subclass of non-TZD insulin-sensitizing agents, have been shown to be peroxisome proliferator-activated receptor (PPAR) gamma agonists. PPARgamma plays a critical role in adipogenesis and PPARgamma agonists have been shown to induce adipocyte differentiation. Here, PPARgamma ligand activity has been assessed in murine 3T3-L1 cells, a commonly used in vitro model of adipogenesis, by measuring their ability to induce adipocyte fatty acid-binding protein (aP2) mRNA expression. In order to perform this task, we have developed a novel, multiwell assay for the direct detection of aP2 mRNA in cell lysates that is based on hybridization of mRNA to target-specific oligonucleotides. These oligonucleotide probes are conjugated to enzymes that efficiently process unique chemical substrates into robust fluorescent products. Ribosomal protein 36B4 mRNA, a gene whose expression is unaffected by adipogenesis, serves as the control in the assay. Two assay formats have been developed, a single analyte assay in which aP2 and 36B4 mRNA expression are assayed in separate lysate aliquots and a dual analyte assay which can measure aP2 and 36B4 mRNA simultaneously. Both forms of the assay have been used to quantify attomole levels of aP2 and 36B4 mRNAs in differentiating 3T3-L1 preadipocytes treated with PPARgamma agonists. The potencies of PPARgamma agonists determined by this novel methodology showed good correlation with those derived from aP2 mRNA slot-blot analysis and PPARgamma transactivation assays. We conclude that the aP2 single and dual analyte assays both provide specific and sensitive measurements of endogenous aP2 mRNA levels that can be used to assess the activity of PPARgamma ligands in 3T3-L1 cells. Since the assay obviates the need for RNA isolation and is performed in an automatable multiwell format, it can serve as a high-throughput, cell-based screen for the identification and characterization of PPARgamma modulators.     

Combination of monocyte-derived dendritic cells and activated T cells which express CD40 ligand: a new approach to cancer immunotherapy

Interactions between dendritic cells (DCs) and activated T cells are critically important for the establishment of an effective immune response. To develop the basis for a new DC-based cancer vaccine, we investigated cell-to-cell interactions between human monocyte-derived DCs and autologous T cells that are activated to express the CD40 ligand (CD40L). Peripheral blood monocytes were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) to induce differentiation of DCs. Activated T cells (ATs) consisted of autologous peripheral blood lymphocytes that had been activated with phytohemagglutinin (PHA) and then stimulated with calcium ionophore to up-regulate expression of CD40L. Coculture of these DCs and ATs induced significant production of interleukin 12 (IL-12) and also enhanced the production of interferon (IFN-). The production of IL-12 was blocked by an anti-CD40L antibody or by separation of the DC and AT fractions by a permeable membrane. Furthermore, coculture of DCs and ATs induced DCs to upregulate CD83 expression and stimulated migration of DCs toward the macrophage inflammatory protein 3- (MIP-3). ATs also migrated toward the MIP-3. These results suggest a combination of DCs and ATs as a potentially effective therapeutic strategy.     

Zippers Make Signals: NCAM-mediated Molecular Interactions and Signal Transduction

The neural cell adhesion molecule, NCAM, is involved in multiple cis- and trans-homophilic interactions (NCAM binding to NCAM) thereby facilitating cell–cell adhesion through the formation of zipper-like NCAM-complexes. NCAM is also involved in heterophilic interactions with a number of proteins and extracellular matrix molecules. Some of these heterophilic interactions are mutually exclusive, and some interfere with or are dependent on homophilic NCAM interactions. Furthermore, both homo- and heterophilic interactions are modulated by posttranslational modifications of NCAM. Heterophilic NCAM-interactions initiate several intracellular signal transduction pathways ultimately leading to biological responses involving cellular differentiation, proliferation, migration and survival. Both homo- and heterophilic NCAM-interactions can be mimicked by synthetic peptides, which can induce NCAM-like signalling, and in vitroand in vivo studies suggest that such NCAM mimetics may be used for the treatment of neurodegenerative disorders.Special issue dedicated to Lawrence F. Eng.