Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INF alpha and INF gamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

Quantum mechanics studies of the tautomers of dlhydrodiacetylformoin, an important Maillard intermediate and the synthesis of furaneol

Conformational analyses have been carried out on the acyclic and cyclic forms of dihydrodiacetylformoin, an important Maillard intermediate and precursor for furaneol. For the acyclic forms, the 2,5-dicarbonyl isomers have the lowest energy, while for the cyclic forms, the 3-carbonyl are favoured over the 4-carbonyl isomers. The likely path for cyclisation is investigated and it is shown that the favoured path is dependent upon the relative chiralities of the carbon atoms and in particular that the reaction proceeds more readily if C2 and C3 have different chiralities. After cyclisation, the reaction path to produce furaneol proceeds via the loss of a water molecule. This reaction has been studied with a model including two water molecules and a hydroxide anion and shows relatively low-energy barriers. (C) 2008 Published by Elsevier B.V.

Dioxadiaza- and trioxadiaza-macrocycles containing one dibenzofuran unit selective for cadmium

The binding properties of dioxadiaza-([17](DBF) N2O2) and trioxadiaza- ([22](DBF)N2O3), macrocyclic ligands containing a rigid dibenzofuran group ( DBF), to metal cations and structural studies of their metal complexes have been carried out. The protonation constants of these two ligands and the stability constants of their complexes with Ca2+, Ba2+, and Mn2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+, were determined at 298.2 K in methanol-water ( 1 : 1, v/v), and at ionic strength 0.10 mol dm(-3) in KNO3. The values of the protonation constants of both ligands are similar, indicating that no cavity size effect is observed. Only mononuclear complexes of these ligands with the divalent metal ions studied were found, and their stability constants are lower than expected, especially for the complexes of the macrocycle with smaller cavity size. However, the Cd2+ complex with [ 17]( DBF) N2O2 exhibits the highest value of stability constant for the whole series of metal ions studied, indicating that this ligand reveals a remarkable selectivity for cadmium(II) in the presence of all the metal ions studied, except copper( II), indicating that this ligand reveals a remarkable selectivity for cadmium( II) in the presence of the mentioned metal ions. The crystal structures of H-2[17](DBF)N2O32+ (diprotonated form of the ligand) and of its cadmium complex were determined by X-ray diffraction. The Cd2+ ion fits exactly inside the macrocyclic cavity exhibiting coordination number eight by coordination to all the donor atoms of the ligand, and additionally to two oxygen atoms from one nitrate anion and one oxygen atom from a water molecule. The nickel( II) and copper( II) complexes with the two ligands were further studied by UV-vis-NIR and the copper( II) complexes also by EPR spectroscopic techniques in solution indicating square-pyramidal structures and suggesting that only one nitrogen and oxygen donors of the ligands are bound to the metal. However an additional weak interaction of the second nitrogen cannot be ruled out.

Ultrasonic chemical oxidative degradations of 1,3-dialkylimidazolium ionic liquids and their mechanistic elucidations

A highly efficient process for oxidative degradation of 1,3-dialkylimidazolium ionic liquids in hydrogen peroxide/acetic acid aqueous medium assisted by ultrasonic chemical irradiation is, for the first time, described. It is shown that more than 93% of the 1,3-dialkylimidazolium cation with the corresponding Cl-, Br-, BF4- and PF6- counter-anions at a concentration of 2.5 mM can be degraded at 50 degrees C within 12 h while at 72 h the conversions approach 99%. A tentative mechanism for the degradation of these ILs is for the first time proposed through a detailed kinetic analysis of several characteristic transients and/or immediate products, which are identified during the ILs degradation using GC-MS. The results clearly indicate that three hydrogen atoms in the imidazolium ring are the first sites preferably oxidized, followed by cleavage of the alkyl groups attached to the N atoms from the ring. The nature of the alkyl chain length on the imidazolium ring and the type of counter anion do not seem to affect the degradation process. Further, selective fragmentations of C-N bonds of the imidazolium or derived ring lead to ring opening, forming degraded intermediates. It is also shown that acetoxyacetic acid and biurea are the final kinetically stable degraded products from the ILs under the degradation conditions.

Halo-derivatised calix[4]tubes

Halo functionalisation of calix[4] tubes has been investigated through both derivatisation of individual calix[4]arenes and calix[4] tubes, using classical synthetic methods, to allow preparation of a series of novel derivatives. The solution and solid state properties are in accordance with the constituent calix[4] arenes adopting flattened cone arrangements which on complexation with potassium simplify to a regular cone. Electrospray and H-1 NMR studies, combined with molecular modelling have been used to ascertain the metal binding of this new series of cryptand like ionophores, demonstrating their retained selectivity for binding potassium over other Group 1 metals and the dependence on counter anion in the weak binding of silver.

Incorporation of a sodium ion guest in the host of copper(II)-Schiff-base complexes: Structural characterization and magnetic study

Three copper(II) complexes, [CuL1], [CuL2] and [CuL3] where L-1, L-2 and L-3 are the tetradentate di-Schiff-base ligands prepared by the condensation of acetylacetone and appropriate diamines (e.g. 1,2-diaminoethane, 1,2-diaminopropane and 1,3-diaminopropane, respectively) in 2:1 ratios, have been prepared. These complexes act as host molecules and include a guest sodium ion by coordinating through the oxygen atoms to result in corresponding new trinuclear complexes, [(CuL1)(2)Na(ClO4)(H2O)][CuL1], [(CuL2)(2)Na(ClO4)(H2O)] (2) and [(CuL3)(2)Na(ClO4)(H2O)] (3) when crystallized from methanol solution containing sodium perchlorate. All three complexes have been characterized by single crystal X-ray crystallography. In all the complexes, the sodium cation has a six-coordinate distorted octahedral environment being bonded to four oxygen atoms from two Schiff-base complexes of Cu(II) in addition to a perchlorate anion and a water molecule. The copper atoms are four coordinate in a square planar environment being bonded to two oxygen atoms and two nitrogen atoms of the Schiff-base ligand. The variable temperature susceptibilities for complexes 1-3 were measured over the range 2-300 K. The isotropic Hamiltonian, H = g(1)beta HS1 + g(2)beta HS2 + J(12)S(1)S(2) + g(3)beta HS3 for complex 1 and H = g(1)beta HS1 + g2 beta HS2 +J(12)S(1)S(2) for complexes 2 and 3 has been used to interpret the magnetic data. The best fit parameters obtained are: g(1) = g(2) = 2.07(0), J = - 1.09(1) cm(-1) for complex 1, g(1) = g(2) = 2.06(0), J = -0.55(1) cm(-1) for complex 2 and g1 = g2 = 2.07(0).J = -0.80(1) cm(-1) for 3. Electrochemical studies displayed an irreversible Cu(II)/Cu(I) one-electron reduction process. (C) 2008 Elsevier Ltd. All rights reserved.

Occurrence of ribbons of cyclic water pentamers in a metallo-organic framework formed by spontaneous fixation of CO2

In the reaction of equimolar amounts of copper(II) acetate with 2,2'-dipyridylamine (DPA) in aqueous tetrahydrofuran, in presence of KOH, aerial CO2 is spontaneously fixed to the carbonate anion yielding [Cu(DPA)(CO3)] . 3H(2)O (1). X-ray crystallography shows the presence of zigzag ribbons of cyclic water pentamers in the channels of a chain-like metallo-organic framework. The water ribbons are stabilised by hydrogen bonds to the metallo-organic backbone. Each (H2O)(5) pentamer is approximately planar.

Synthesis, reactivity, and X-ray crystal structure of some mixed-ligand oxovanadium(V) complexes: first report of binuclear oxovanadium(V) complexes containing 4,4 '-Bipyridine type bridge

Reaction of the tridentate ONO Schiff-base ligand 2-hydroxybenzoylhydrazone of 2-hydroxybenzoylhydrazine (H2L) with VO(acac)(2) in ethanol medium produces the oxoethoxovanadium(V) complex [VO(OEt)L] (A), which reacts with pyridine to form [VO(OEt)L center dot(py)] (1). Complex 1 is structurally characterized. It has a distorted octahedral O4N2 coordination environment around the V(V) acceptor center. Both complexes A and 1 in ethanol medium react with neutral monodentate Lewis bases 2-picoline, 3-picoline, 4-picoline, 4-amino pyridine, imidazole, and 4-methyl imidazole, all of which are stronger bases than pyridine, to produce dioxovanadium(V) complexes of general formula BH[VO2L]. Most of these dioxo complexes are structurally characterized, and the complex anion [VO2L](-) is found to possess a distorted square pyramidal structure. When a solution/suspension of a BH[VO2L] complex in an alcohol (ROH) is treated with HCl in the same alcohol, it is converted into the corresponding monooxoalkoxo complex [ O(OR)L], where R comes from the alcohol used as the reaction medium. Both complexes A and 1 produce the 4,4'-bipyridine-bridged binuclear complex [VO(OEt)L](2)(mu-4,4'-bipy) (2), which, to the best of our knowledge, represents the first report of a structurally characterized 4,4'-bipyridine-bridged oxovanadium(V) binuclear complex. Two similar binuclear oxovanadium(V) complexes 3 and 4 are also synthesized and characterized. All these binuclear complexes (2-4), on treatment with base B, produce the corresponding mononuclear dioxovanadium(V) complexes (5-10).

Free radicals, metals and antioxidants in oxidative stress-induced cancer

Oxygen-free radicals, more generally known as reactive oxygen species (ROS) along with reactive nitrogen species (RNS) are well recognised for playing a dual role as both deleterious and beneficial species. The "two-faced" character of ROS is substantiated by growing body of evidence that ROS within cells act as secondary messengers in intracellular signalling cascades, which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. The cumulative production of ROS/RNS through either endogenous or exogenous insults is termed oxidative stress and is common for many types of cancer cell that are linked with altered redox regulation of cellular signalling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (8-OH-G) have been noted in various tumours, strongly implicating such damage in the etiology of cancer. It appears that the DNA damage is predominantly linked with the initiation process. This review examines the evidence for involvement of the oxidative stress in the carcinogenesis process. Attention is focused on structural, chemical and biochemical aspects of free radicals, the endogenous and exogenous sources of their generation, the metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel)-mediated formation of free radicals (e.g. Fenton chemistry), the DNA damage (both mitochondrial and nuclear), the damage to lipids and proteins by free radicals, the phenomenon of oxidative stress, cancer and the redox environment of a cell, the mechanisms of carcinogenesis and the role of signalling cascades by ROS; in particular. ROS activation of AP-1 (activator protein) and NF-kappa B (nuclear factor kappa B) signal transduction pathways, which, in turn lead to the transcription of genes involved in cell growth regulatory pathways. The role of enzymatic (superoxide dismutase (Cu. Zn-SOD. Mn-SOD), catalase, glutathione peroxidase) and non-enzymatic antioxidants (Vitamin C, Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic acid), flavonoids, selenium and others) in the process of careinogenesis as well as the antioxidant interactions with various regulatory factors, including Ref-1, NF-kappa B, AP-1 are also reviewed. 2006 Elsevier Ireland Ltd. All rights reserved.

Ditopic redox-active polyferrocenyl zinc(II) dithiocarbamate macrocyclic receptors: synthesis, coordination and electrochemical recognition properties

The synthesis of a range of ditopic polyferrocenyl zinc(II) dithiocarbamate macrocyclic receptors containing ferrocene groups on the macrocycle's periphery and/or as part of the cyclic cavity is reported. The assemblies have been characterised by a range of spectroscopic techniques, electrochemical studies and in two cases by X-ray structure determination. The ability of these host systems to bind and sense electrochemically anionic guest species, isonicotinate and benzoate, and neutral 4-picoline guest was examined by H-1 NMR and cyclic voltammetric titration studies. The strongest association was found between the isonicotinate anion and a dinuclear zinc(II) receptor whose macrocyclic cavity is of complementary size to complex this bidentate guest species in a cooperative manner. Cyclic voltammetric studies demonstrated that all receptors can electrochemically sense the binding of isonicotinate and benzoate via significant cathodic perturbations of the respective ferrocene redox couple.

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1(wt)) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1(wt) in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1(wt) expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [H-3]D-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1(wt). The hSOD1-induced decline in GLT-1 protein and [H-3]D-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1(wt) in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.

Influence of apolipoprotein E genotype and dietary alpha-tocopherol on redox status and C-reactive protein levels in apolipoprotein E3 and E4 targeted replacement mice

The molecular basis of the positive association between apoE4 genotype and CVD remains unclear. There is direct in vitro evidence indicating that apoE4 is a poorer antioxidant relative to the apoE3 isoform, with some indirect in vivo evidence also available. Therefore it was hypothesised that apoE4 carriers may benefit from alpha-tocopherol (alpha-Toc) supplementation. Targeted replacement mice expressing the human apoE3 and apoE4 were fed with a diet poor (0 mg/kg diet) or rich (200 mg/kg diet) in alpha-Toc for 12 weeks. Neither apoE genotype nor dietary alpha-Toc exerted any effects on the antioxidant defence system, including glutathione, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities. In addition, no differences were observed in mitogen-induced lymphocyte proliferation. alpha-Toc concentrations were modestly higher in plasma and lower in tissues of apoE4 compared with apoE3 mice, with the greatest differences evident in the lung, suggesting that an apoE4 genotype may reduce alpha-Toc delivery to tissues. A tendency towards increased plasma F-2-isoprostanes in apoE4 mice was observed, while liver thiobarbituric acid-reactive substances did not differ between apoE3 and apoE4 mice. In addition, C-reactive protein (CRP) concentrations were reduced in apoE4 mice indicating that this positive effect on CRP may in part negate the increased CVD risk associated with an apoE4 genotype.

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 mu L/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 mu M). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 mu L/mL WCE, 3.7-fold 1 mu M PEITC) and SOD2 (12.1-fold, 10 mu L/mL WCE, 7.3-fold, 10 mu M PEITC), and increased SOD2 activity (1.9-fold, 10 mu L/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.