Crystal structures of Λ-[Ru(phen)2dppz]2+ with oligonucleotides containing TA/TA and AT/AT steps show two intercalation modes

The ruthenium complex [Ru(phen)2(dppz)] (where phen is a phenanthroline and dppz a dipyridyl–phenazine ligand) is known as a ‘light switch’ complex because its luminescence in solution is significantly enhanced in the presence of DNA. This property is poised to serve in diagnostic and therapeutic applications, but its binding mode with DNA needs to be elucidated further. Here, we describe the crystal structures of the L enantiomer bound to two oligonucleotide duplexes. The dppz ligand intercalates symmetrically and perpendicularly from the minor groove of the d(CCGGTACCGG)2 duplex at the central TA/TA step, but not at the central AT/AT step of d(CCGGATCCGG)2. In both structures, however, a second ruthenium complex links the duplexes through the combination of a shallower angled intercalation into the C1C2/G9G10 step at the end of the duplex, and semi-intercalation into the G3G4 step of an adjacent duplex. The TA/TA specificity of the perpendicular intercalation arises from the packing of phenanthroline ligands against the adenosine residue.

Variability in fecal water genotoxicity, determined using the Comet assay, is independent of endogenous N-nitroso compound formation attributed to red meat consumption

Red meat consumption causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC). The genotoxic effects of these ATNCs were investigated using two different Comet assay protocols to determine the genotoxicity of fecal water samples. Fecal water samples were obtained from two studies of a total of 21 individuals fed diets containing different amounts of red meat, protein, heme, and iron. The first protocol incubated the samples with HT-29 cells for 5 min at 4 degrees C, whereas the second protocol used a longer exposure time of 30 min and a higher incubation temperature of 37 degrees C. DNA strand breaks were quantified by the tail moment (DNA in the comet tail multiplied by the comet tail length). The results of the two Comet assay protocols were significantly correlated (r = 0.35, P = 0.003), however, only the second protocol resulted in detectable levels of DNA damage. Inter-individual effects were variable and there was no effect on fecal water genotoxicity by diet (P > 0.20), mean transit time (P = 0.588), or weight (P = 0.705). However, there was a highly significant effect of age (P = 0.019). There was no significant correlation between concentrations of ATNCs in fecal homogenates and fecal water genotoxicity (r = 0.04, P = 0.74). ATNC levels were lower in fecal water samples (272 microg/kg) compared to that of fecal homogenate samples (895 microg/kg) (P < 0.0001). Failure to find dietary effects on fecal water genotoxicity may therefore be attributed to individual variability and low levels of ATNCs in fecal water samples.

The effect of copper(II), iron(II) sulphate and vitamin C combinations on the weak antimicrobial activity of (+)-catechin against Staphylococcus aureus and other microbes

Few attempts have been made to improve the activity of plant compounds with low antimicrobial efficacy. (+)-Catechin, a weak antimicrobial tea flavanol, was combined with putative adjuncts and tested against different species of bacteria. Copper(II) sulphate enhanced (+)-catechin activity against Pseudomonas aeruginosa but not Staphylococcus aureus, Proteus mirabilis or Escherichia coli. Attempts to raise the activity of (+)-catechin against two unresponsive species, S. aureus and E. coli, with iron(II) sulphate, iron(III) chloride, and vitamin C, showed that iron(II) enhanced (+)-catechin against S. aureus, but not E. coli; neither iron(III) nor combined iron(II) and copper(II), enhanced (+)-catechin activity against either species. Vitamin C enhanced copper(II) containing combinations against both species in the absence of iron(II). Catalase or EDTA added to active samples removed viability effects suggesting that active mixtures had produced H2O2via the action of added metal(II) ions. H2O2 generation by (+)-catechin plus copper(II) mixtures and copper(II) alone could account for the principal effect of bacterial growth inhibition following 30 minute exposures as well as the antimicrobial effect of (+)-catechin–iron(II) against S. aureus. These novel findings about a weak antimicrobial flavanol contrast with previous knowledge of more active flavanols with transition metal combinations. Weak antimicrobial compounds like (+)-catechin within enhancement mixtures may therefore be used as efficacious agents. (+)-Catechin may provide a means of lowering copper(II) or iron(II) contents in certain crop protection and other products.

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.

Robotic multiwell planar patch-clamp for native and primary mammalian cells

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.

Host–guest supramolecular interactions in the coordination compounds of 4,4′-azobis(pyridine) with MnX2(X = NCS–, NCNCN–, and PF6–): structural analyses and theoretical study

Three new Mn(II) coordination compounds {[Mn(NCNCN)2(azpy)]·0.5azpy}n (1), {[Mn(NCS)2(azpy)(CH3OH)2]·azpy}n (2), and [Mn(azpy)2(H2O)4][Mn(azpy)(H2O)5]·4PF6·H2O·5.5azpy (3) (where azpy = 4,4'-azobis-(pyridine)) have been synthesized by self-assembly of the primary ligands, dicyanamide, thiocyanate, and hexafluorophosphate, respectively, together with azpy as the secondary spacer. All three complexes were characterized by elemental analyses, IR spectroscopy, thermal analyses, and single crystal X-ray crystallography. The structural analyses reveal that complex 1 forms a two-dimensional (2D) grid sheet motif These sheets assemble to form a microporous framework that incorporates coordination-free azpy by host-guest pi center dot center dot center dot pi. and C-H center dot center dot center dot N hydrogen bonding interactions. Complex 2 features azpy bridged one-dimensional (ID) chains of centrosymmetric [Mn(NCS)(2)(CH3OH)(2)} units which form a 2D porous sheet via a CH3 center dot center dot center dot pi supramolecular interaction. A guest azpy molecule is incorporated within the pores by strong H-bonding interactions. Complex 3 affords a 0-D motif with two monomeric Mn(II) units in the asymmetric unit. There exist pi center dot center dot center dot pi, anion center dot center dot center dot pi, and strong hydrogen bonding interactions between the azpy, water, and the anions. Density functional theory (DFT) calculations, at the M06/6-31+G* level of theory, are used to characterize a great variety of interactions that explicitly show the importance of host-guest supramolecular interactions for the stabilization of coordination compounds and creation of the fascinating three-dimensional (3D) architecture of the title compounds.

Immunomodulatory effects of a probiotic drink containing Lactobacillus casei Shirota in healthy older volunteers

PURPOSE: There is growing evidence that probiotics confer health benefits to the host by modulating immune function, especially in older people, where immunosenescence is a feature even of healthy ageing. The aim of this study was to investigate the effect of a probiotic drink containing Lactobacillus casei Shirota (LcS) on immune function in a healthy non-immunocompromised older population. METHODS: Thirty healthy old volunteers were recruited into a randomized placebo-controlled, single-blind crossover study. The volunteers were supplemented with the probiotic drink containing 1.3 × 10(10) CFU LcS or skimmed milk per day for 4 weeks, followed by 4 weeks of washout and were crossed over to the other treatment. Peripheral blood and saliva samples were collected at baseline and end of each treatment. RESULTS: Probiotic consumption was associated with a significant increase in natural killer (NK) cell activity relative to baseline and a significant decrease in the mean fluorescence intensity of CD25 expression in the resting T cells compared with placebo. Additionally, there was a trend towards an increased ratio of IL-10 to IL-12 relative to baseline after LcS intake. CONCLUSIONS: Consumption of a probiotic drink containing LcS improved NK cell activity and tended to produce a more anti-inflammatory cytokine profile in an older population.

Contribution of non-volatile and volatile compounds to the umami taste and overall flavour of shiitake mushroom extracts and their application as flavour enhancers in cooked minced meat

Aqueous extracts of dried shiitake mushrooms (Lentinus edodes) were prepared as taste and flavour enhancers for meat formulations. Effects of time and temperature on the chemical and sensory properties of the extracts were examined. Extracts prepared at 70 °C had significantly higher concentrations (p<0.001) of the savoury tasting 5’-ribonucleotides than those prepared at 22 °C but increasing the extraction time from 30 to 360 mins only increased their recovery slightly (p=0.053). In contrast, higher temperature extracts had considerably smaller concentrations of the major volatile compounds, such as lenthionine, 1-octen-3-ol, 1,3-dithiethane and dimethyl disulfide, because of loss through volatilisation. A sensory discrimination test showed that the lower temperature extract was perceived to have less umami taste than the higher temperature extract (p=0.048). Incorporating the 70 °C shiitake extract into minced meat formulations led to significantly higher levels of savoury tasting 5’-ribonucleotides in the cooked meat but no significant difference in umami perception.

Daidzein, R-(+)equol and S-(-)equol inhibit the invasion of MDA-MB-231 breast cancer cells potentially via the down-regulation of matrix metalloproteinase-2

PURPOSE: Soy isoflavones may inhibit tumor cell invasion and metastasis via their effects on matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The current study investigates the effects of daidzein, R- and S-equol on the invasion of MDA-MB-231 human breast cancer cells and the effects of these compounds on MMP/TIMP expression at the mRNA level. METHODS: The anti-invasive effects of daidzein, R- and S-equol (0, 2.5, 10, 50 μM) on MDA-MB-231 cells were determined using the Matrigel invasion assay following 48-h exposure. Effects on MMP-2, MMP-9, TIMP-1 and TIMP-2 expression were assessed using real-time PCR. Chiral HPLC analysis was used to determine intracellular concentrations of R- and S-equol. RESULTS: The invasive capacity of MDA-MB-231 cells was significantly reduced (by approximately 50-60 %) following treatment with 50 μM daidzein, R- or S-equol. Anti-invasive effects were also observed with R-equol at 2.5 and 10 μM though overall equipotent effects were induced by all compounds. Inhibition of invasion induced by all three compounds at 50 μM was associated with the down-regulation of MMP-2, while none of the compounds tested significantly affected the expression levels of MMP-9, TIMP-1 or TIMP-2 at this concentration. Following exposure to media containing 50 μM R- or S-equol for 48-h intracellular concentrations of R- and S-equol were 4.38 ± 1.17 and 3.22 ± 0.47 nM, respectively. CONCLUSION: Daidzein, R- and S-equol inhibit the invasion of MDA-MB-231 human breast cancer cells in part via the down-regulation of MMP-2 expression, with equipotent effects observed for the parent isoflavone daidzein and the equol enantiomers.

Effect of forage conservation method on plasma lipids, mammary lipogenesis, and milk fatty acid composition in lactating cows fed diets containing a 60:40 forage-to-concentrate ratio

The effects of forage conservation method on plasma lipids, mammary lipogenesis, and milk fat were examined in 2 complementary experiments. Treatments comprised fresh grass, hay, or untreated (UTS) or formic acid treated silage (FAS) prepared from the same grass sward. Preparation of conserved forages coincided with the collection of samples from cows fed fresh grass. In the first experiment, 5 multiparous Finnish Ayrshire cows (229 d in milk) were used to compare a diet based on fresh grass followed by hay during 2 consecutive 14-d periods, separated by a 5-d transition during which extensively wilted grass was fed. In the second experiment, 5 multiparous Finnish Ayrshire cows (53 d in milk) were assigned to 1 of 2 blocks and allocated treatments according to a replicated 3 × 3 Latin square design, with 14-d periods to compare hay, UTS, and FAS. Cows received 7 or 9 kg/d of the same concentrate in experiments 1 and 2, respectively. Arterial concentrations of triacylglycerol (TAG) and phospholipid were higher in cows fed fresh grass, UTS, and FAS compared with hay. Nonesterified fatty acid (NEFA) concentrations and the relative abundance of 18:2n-6 and 18:3n-3 in TAG of arterial blood were also higher in cows fed fresh grass than conserved forages. On all diets, TAG was the principle source of fatty acids (FA) for milk fat synthesis, whereas mammary extraction of NEFA was negligible, except during zero-grazing, which was associated with a lower, albeit positive calculated energy balance. Mammary FA uptake was higher and the synthesis of 16:0 lower in cows fed fresh grass than hay. Conservation of grass by drying or ensiling had no influence on mammary extraction of TAG and NEFA, despite an increase in milk fat secretion for silages compared with hay and for FAS than UTS. Relative to hay, milk fat from fresh grass contained lower 12:0, 14:0, and 16:0 and higher S3,R7,R11,15-tetramethyl-16:0, cis-9 18:1, trans-11 18:1, cis-9,trans-11 18:2, 18:2n-6, and 18:3n-3 concentrations. Even though conserved forages altered mammary lipogenesis, differences in milk FA composition were relatively minor, other than a higher enrichment of S3,R7,R11,15-tetramethyl-16:0 in milk from silages compared with hay. In conclusion, differences in milk fat composition on fresh grass relative to conserved forages were associated with a lower energy balance, increased uptake of preformed FA, and decreased synthesis of 16:0 de novo in the mammary glands, in the absence of alterations in stearoyl-coenzyme A desaturase activity.

Arrays of chiral nanotubes and a layered coordination polymer containing gallium-sulfide supertetrahedral clusters

We report here a unique chiral hybrid gallium sulfide, [NC2H8]2[Ga10S16(N2C12H12)(NC2H7)2] 1, consisting of helical chains of organically-functionalised supertetrahedral clusters which form quadruple-stranded helical nanotubes of ca. 3 nm diameter. This material therefore consists of discrete metal-organic nanotubes which, to the best of our knowledge, are extremely rare. Whilst solvothermal reactions involving 1,2-di(4-pyridyl)ethylene (DPE) resulted in the formation of such single-walled chiral nanotubes, the use of longer 4,4’-trimethylenedipyridine (TMP) ligands resulted in the synthesis of a two-dimensional hybrid gallium sulfide, [C5H6N]3[Ga10S16(OH)(N2C13H14)] 2 in which, for the first time, inorganic and organic linkages between supertetrahedral clusters coexist.

A copper-containing oxytelluride as a promising thermoelectric material for waste heat recovery

The new thermoelectric material BiOCuTe exhibits an electrical conductivity of 224 S cm-1 and a Seebeck coefficient of +186 μV K-1 at 373 K, together with an extremely low lattice thermal conductivity of ∼ 0.5 W m-1 K-1. This results in a ZT of 0.42 at 373 K, which increases to 0.66 at the maximum temperature investigated, 673 K.

Radiative forcing and global warming potentials of 11 halogenated compounds

Radiative forcing values have been calculated for 11 halogenated compounds which are in current use or which have been suggested as possible replacements for the chlorofluorocarbons. Absorption cross-sections measured over a range of atmospheric temperature and pressure conditions as part of a multi-laboratory programme have been used together with a narrow band radiative transfer model. We provide a “best estimate” radiative forcing taking into account the likely vertical profile of the gas in each case. The Global Warming Potential over a variety of time horizons has also been calculated where the lifetime is available. We present the first such information for 1,2-dichloroethane. For chloroform our radiative forcing is 5 times higher than the value used in previous assessments, possibly because these ignored the effect of absorption outside the 800–1200 cm−1 “window”. For several of the other compounds considered here, our forcing is between 10 and 30% lower than previous assessments. The perfluorocarbons have been found to have large global warming potentials, many times that of CFC-11, due to both strong absorption and long lifetimes. The importance of absorption features at wavenumbers below 800 cm−1 and the effect of temperature variations in absorption cross-section on the radiative forcing are also investigated.

Enhancement in corneal permeability of riboflavin using calcium sequestering compounds

Ethylenediaminetetraacetic acid, ethylenediamine-N,N′-disuccinic acid and ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid are polyaminocarboxylic acids that are able to sequester metal ions. Calcium is implicated in maintenance of intercellular matrix, zonula occludens (tight junctions) and zonula adherens of epithelium and endothelium cells. Corneal epithelium is impervious to many aqueous formulations due to it being lipophilic, whereby transcellular drug transit is resisted, whilst tight junctions restrict access via the paracellular route. Research has shown that integrity of tight junctions breaks down through loss of Ca2+ for endothelial and epithelial cells. This study investigates different Ca2+ sequestering compounds and their effect on corneal permeability of riboflavin at physiological pH. Riboflavin is a topically administered ocular drug applied during UV-induced corneal cross-linking for the treatment of keratoconus.