Protease-activated receptor 2, dipeptidyl peptidase I, and proteases mediate Clostridium difficile toxin A enteritis

BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. RESULTS: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Bone morphogenetic proteins (BMP)-4,-6, and-7 potently suppress basal and luteinizing hormone-induced androgen production by bovine theca interna cells in primary culture: Could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and - 7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, - 6, and - 7 potently suppress both basal ( P < 0.0001; respective IC50 values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced ( P < 0.0001; respective IC50 values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3 beta-hydroxysteroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment ( P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and - 7, respectively, and the observation that both antagonists enhanced ( P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP- 4 and - 7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.

A fluxional (CuN2O2)-N-I core: binding of a keto oxygen atom to Cu-I and Ag-I

[Cu(2-acetylpyridine)(2)]ClO4 (1), characterised here, has a novel Cu'N202 core in the solid state. Variable-temperature H-1 NMR studies show that the two chelate rings open up in solution at room temperature and the keto oxygen atoms dangle freely. As the temperature is lowered, the 0 atoms tend to bind to the metal atom. The corresponding silver(I) complex, [Ag(2-acetylpyridine)2]ClO4 (4), characterised by single-crystal X-ray crystallography, has an (AgN2)-N-I core in the solid state as well as in solution. Thus, while 1 is fluxional, 4 is not. In cyclic voltammetry, complex 1 displays a quasireversible Cu-II/I couple with a half-wave potential of 0.40 V vs. SCE. Complex I is easily oxidised by air and H2O2 in methanol to give rise to a dinuclear copper(II) complex where the ligand framework is not simple acetylpyridine. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005).

Biomimetic approach to Galbulimina type I alkaloids

On treatment with trifluoroacetic acid the tetraene precursor 23 underwent Boc deprotection, condensation and an iminium ion accelerated intramolecular Diels-Alder cycloaddition resulting in an iminium species 12, which was further converted into himbacine 1, himbeline 3 and himandravine 4, three out of four Galbulimina type I alkaloids thus providing strong evidence for the proposed biogenesis of this important family of alkaloids.

Amyloid-like fibrillogenesis through supramolecular helix-mediated self-assembly of tetrapeptides containing non-coded alpha-aminoisobutyric acid (Aib) and 3-aminobenzoic acid (m-ABA)

Single-crystal X-ray diffraction studies of two terminally protected tetrapeptides Boc-Ile-Aib-Val-m-ABA-OMe (I) and Boc-Ile-Aib-Phe-m-ABA-OMe (II) (Aib = alpha-aminoisobutyric acid; m-ABA = meta-aminobenzoic acid) reveal that they form continuous H-bonded helices through the association of double-bend (type III and I) building blocks. NMR Studies support the existence of the double-bend (type Ill and I) structures of the peptides in solution also. Field emission scanning electron-microscopic (FE-SEM) and high-resolution transmission electron-microscopic (HR-TEM) images of the peptides exhibit amyloid-like fibrils in the solid state. The Congo red-stained fibrils of peptide I and II, observed between crossed polarizers, show green-gold birefringence, a characteristic of amyloid fibrils.

A hydrogen-1 nuclear magnetic resonance, mass spectral and extended Hückel study of some olefin complexes of rhodium(I) and iridium(I) and the crystal and molecular structure of η4-2,4-dimethylpenta-1,4-diene(η5-formylcyclopentadienyl)rhodium(I)

A 1H NMR study of monosubstituted η-cyclopentadienyl-rhodium(I) complexes of type LLRh(C5H4X) and -iridium(I) complexes of type L2Ir(C5H4X) (L = ethene, LL = 1,3- or 1,5-diolefin; X = C(C6H5)3, CHO, or COOCH3) has been carried out. For complexes of both metals in which the neutral ligand is ethene or a non-conjugated diolefin the NMR spectra of the cyclopentadienyl protons are unusual in that H(2), H(5) resonate to high field either at room temperature or below. The corresponding NMR spectra for the cyclopentadienyl ring protons of complexes where the neutral ligand is a conjugated diene are, with one exception, normal. A single crystal X-ray structural analysis of (η4-2,4-dimethylpenta-1,4-diene)(η5-formylcyclopentadienyl)rhodium(I) (which exhibits an abnormal 1H NMR spectrum) reveals substantial localisation of electron density in the C(3)C(4) Cp ring bond (1.283(33) Å) which may be consistent with a contribution from an ‘allyl-ene’ rotamer to the ring—metal bonding scheme. An extended Hückel calculation with self consistent charge iteration was performed on this complex. The results predict a greater Mulliken overlap population for the C(3)C(4) bond in the cyclopentadienyl ring and show that the localisation is dependent on both the Cp ring substituent and the nature of the diolefin. The mass spectral fragmentation patterns of some representative diene complexes of iridium(I) and rhodium(I) are presented.

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.

Moving towards a more mechanistic approach in the determination of soil heat flux from remote measurements - II. Diurnal shape of soil heat flux

For vegetated surfaces, calculation of soil heat flux, G, with the Exact or Analytical method requires a harmonic analysis of below-canopy soil surface temperature, to obtain the shape of the diurnal course of G. When determining G with remote sensing methods, only composite (vegetation plus soil) radiometric brightness temperature is available. This paper presents a simple equation that relates the sum of the harmonic terms derived for the composite radiometric surface temperature to that of belowcanopy soil surface temperature. The thermal inertia, Gamma(,) for which a simple equation has been presented in a companion paper, paper I, is used to set the magnitude of G. To assess the success of the method proposed in this paper for the estimation of the diurnal shape of G, a comparison was made between 'remote' and in situ calculated values from described field sites. This indicated that the proposed method was suitable for the estimation of the shape of G for a variety of vegetation types and densities. The approach outlined in paper I, to obtain Gamma, was then combined with the estimated harmonic terms to predict estimates of G, which were compared to values predicted by empirical remote methods found in the literature. This indicated that the method proposed in the combination of papers I and II gave reliable estimates of G, which, in comparison to the other methods, resulted in more realistic predictions for vegetated surfaces. This set of equations can also be used for bare and sparsely vegetated soils, making it a universally applicable method. (C) 2007 Elsevier B.V. All rights reserved.

The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines

Viral fusion proteins mediate the merger of host and viral membranes during cell entry for all enveloped viruses. Baculovirus glycoprotein gp64 (gp64) is unusual in promoting entry into both insect and mammalian cells and is distinct from established class I and class II fusion proteins. We report the crystal structure of its postfusion form, which explains a number of gp64's biological properties including its cellular promiscuity, identifies the fusion peptides and shows it to be the third representative of a new class (III) of fusion proteins with unexpected structural homology with vesicular stomatitis virus G and herpes simplex virus type 1 gB proteins. We show that domains of class III proteins have counterparts in both class I and II proteins, suggesting that all these viral fusion machines are structurally more related than previously thought.

Helices, chirality and interpenetration: the versatility and remarkable interconversion of silver-copper cyanide frameworks

The structural transformations between cesium silver-copper cyanides under modest conditions, both in solution and in the solid state, are described. Three new cesium silver(I) copper(I) cyanides with three-dimensional (3-D) framework structures were prepared as single crystals from a one-pot reaction initially heated under hydrothermal conditions. The first product to appear, Cs3Ag2Cu3(CN)(8) (I), when left in contact with the supernatant produced CsAgCu(CN)(3) (II) and CsAgCu(CN)(3)center dot 1/3H(2)O (III) over a few months via a series of thermodynamically controlled cascade reactions. Crystals of the hydrate (III) can be dehydrated to polycrystalline CsAgCu(CN)(3) (II) on heating at 100 degrees C in a remarkable solid-state transformation involving substantial breaking and reconnection of metal-cyanide linkages. Astonishingly, the conversion between the two known polymorphs of CsAg2Cu(CN)(4), which also involves a major change in connectivity and topology, occurs at 180 degrees C as a single-crystal to single-crystal transformation. Structural features of note in these materials include the presence of helical copper-cyanide chains in (I) and (II), which in the latter compound produce a chiral material. In (II) and (III), the silver-copper cyanide networks are both self- and interpenetrating, features also seen in the known polymorphs of CsAg2Cu(CN)(4).

beta-Sheet mediated self-assembly of dipeptides of omega-amino acids and remarkable fibrillation in the solid state

Single crystal X-ray diffraction studies show that the extended structure of dipeptide I Boc-beta-Ala-m-ABA-OMe (m-ABA: meta-aminobenzoic acid) self-assembles in the solid state by intermolecular hydrogen bonding to create an infinite parallel P-sheet structure. In dipeptide II Boc-gamma-Abu-m-ABA-OMe (gamma-Abu: gamma-aminobutyric acid), two such parallel beta-sheets are further cross-linked by intermolecular hydrogen bonding through m-aminobenzoic acid moieties. SEM (scanning electron microscopy) studies reveal that both the peptides I and II form amyloid-like fibrils in the solid state. The fibrils are also found to be stained readily by Congo red, a characteristic feature of the amyloid fiber whose accumulation causes several fatal diseases such as Alzheimer's, prion-protein etc.

m-aminobenzoic acid inserted beta-turn in acyclic tripeptides: a peptidomimetic design

X-ray diffraction studies show that peptides Boc-Leu-Aib-m-ABA-OMe (I) (Aib, alpha-aminoisobutyric acid; m-ABA, meta-aminobenzoic acid) and Boc-Phe-Aib-m-ABA-OMe, (II) adopt a type-II beta-turn conformation, solely stabilized by co-operative steric interactions amongst the amino acid residues. This type of U-turn without any intramolecular hydrogen bonding is generally referred to as an open turn. Although there are some examples of constrained cyclic peptides in which o-substituted benzenes have been inserted to mimic the turn region of the neurotrophin, a nerve growth factor, peptides I and II present novel two examples where m-aminobenzoic acid has been incorporated in the beta-turn of acyclic tripeptides. The result also demonstrates the first crystallographic evidence of a beta-turn structure containing an inserted m-aminobenzoic acid, which can be considered as a rigid gamma-aminobutyric acid with an all-trans extended configuration. (c) 2005 Elsevier Ltd. All rights reserved.

Design of a new foldamer turn-linker-turn in acyclic hexapeptides and formation of channels through self-assembly

Single crystal X-ray diffraction studies and solvent dependent NMR titration reveal that the designed pepticles I and 11, Boc-Xx(1)-Aib(2)-Yy(3)-NH(CH2)(2)NH-Yy(3)-Aib(2)-Xx(1)-Boc, where Xx and Yy are lie and Leu in peptide I and Leu and Val in peptide 11, respectively, fold into a turn-linker-turn (T-L-T) conformation both in the solid state and in solution. In the crystalline state the T-L-T foldamers; of peptide I and II self-assemble to form a three-dimensional framework of channels. The insides of the channels are hydrophilic and found to contain solvent CHCl3 hydrogen bonded to exposed C=O of Aib located at the turn regions. (c) 2008 Elsevier B.V. All rights reserved.

Extraction and coordination studies of the unexplored bifunctional ligand carbamoyl methyl sulfoxide (CMSO) with uranium(VI) and cerium(III) nitrates. Synthesis and structures of [UO2(NO3)(2)((PhSOCH2CONBu2)-Bu-i)] and [Ce(NO3)(3)(PhSOCH2CONBu2)(2)]

The bifunctional carbamoyl methyl sulfoxide ligands, PhCH2SOCH2CONHPh (L-1), PhCH2SOCH2CONHCH2Ph (L-2), (PhSOCH2CONPr2)-Pr-i (L-3), PhSOCH2CONBu2 (L-4), (PhSOCH2CONBu2)-Bu-i (L-5) and PhSOCH2CON(C8H17)(2) (L-6) have been synthesized and characterized by spectroscopic methods. The selected coordination chemistry of L-1, L-3, L-4 and L-5 with [UO2(NO3)(2)] and [Ce(NO3)(3)] has been evaluated. The structures of the compounds [UO2(NO3)(2)((PhSOCH2CONBu2)-Bu-i)] (10) and [Ce(NO3)(3)(PhSOCH2CONBu2)(2)] (12) have been determined by single crystal X-ray diffraction methods. Preliminary extraction studies of ligand L-6 with U(VI), Pu(IV) and Am(III) in tracer level showed an appreciable extraction for U(VI) and Pu(IV) in up to 10 M HNO3 but not for Am(III). Thermal studies on compounds 8 and 10 in air revealed that the ligands can be destroyed completely on incineration. The electron spray mass spectra of compounds 8 and 10 in acetone show that extensive ligand distribution reactions occur in solution to give a mixture of products with ligand to metal ratios of 1 : 1 and 2 : 1. However, 10 retains its solid state structure in CH2Cl2.

The syntheses, structures and redox properties of phosphine-gold(I) and triruthenium-carbonyl cluster derivatives of tolans

The syntheses of several ethynyl-gold(I) phosphine substituted tolans (1,2-diaryl acetylenes) of general form [Au(C=CC6H4C=CC6H4X)(PPh3)] are described [X = Me (2a), OMe (2b), CO2Me (2c), NO2 (2d), CN (2e)]. These complexes react readily with [Ru-3(CO) 10(mu-dppm)] to give the heterometallic clusters [Ru3(mu-AuPPh3)(mu-eta(1), eta(2)-C2C6H4C, CC6H4X)(CO)(7)(mu-dppm)] (3a-e). The crystallographically determined molecular structures of 2b, 2d, 2e and 3a-e are reported here, that of 2a having been described on a previous occasion. Structural, spectroscopic and electrochemical studies were conducted and have revealed little electronic interaction between the remote substituent and the organometallic end-caps. (C) 2007 Elsevier B. V. All rights reserved.