Molecular detection of exercise-induced free radicals following ascorbate prophylaxis in type 1 diabetes mellitus: a randomised controlled trial

Aims/hypothesis: Patients with type 1 diabetes mellitus are more susceptible than healthy individuals to exercise-induced oxidative stress and vascular endothelial dysfunction, which has important implications for the progression of disease. Thus, in the present study, we designed a randomised double-blind, placebo-controlled trial to test the original hypothesis that oral prophylaxis with vitamin C attenuates rest and exercise-induced free radical-mediated lipid peroxidation in type 1 diabetes mellitus. Methods: All data were collected from hospitalised diabetic patients. The electron paramagnetic resonance spectroscopic detection of spin-trapped a-phenyl-tert-butylnitrone (PBN) adducts was combined with the use of supporting markers of lipid peroxidation and non-enzymatic antioxidants to assess exercise-induced oxidative stress in male patients with type 1 diabetes (HbA1c 7.9±1%, n=12) and healthy controls (HbA1c 4.6±0.5%, n=14). Following participant randomisation using numbers in a sealed envelope, venous blood samples were obtained at rest, after a maximal exercise challenge and before and 2 h after oral ingestion of 1 g ascorbate or placebo. Participants and lead investigators were blinded to the administration of either placebo or ascorbate treatments. Primary outcome was the difference in changes in free radicals following ascorbate ingestion. Resuts: Six diabetic patients and seven healthy control participants were randomised to each of the placebo and ascorbate groups. Diabetic patients (n=12) exhibited an elevated concentration of PBN adducts (p<0.05 vs healthy, n=14), which were confirmed as secondary, lipid-derived oxygen-centred alkoxyl (RO•) radicals (a nitrogen=1.37 mT and aßhydrogen=0.18 mT). Lipid hydroperoxides were also selectively elevated and associated with a depression of retinol and lycopene (p<0.05 vs healthy). Vitamin C supplementation increased plasma vitamin C concentration to a similar degree in both groups (p<0.05 vs pre-supplementation) and attenuated the exercise-induced oxidative stress response (p<0.05 vs healthy). There were no selective treatment differences between groups in the primary outcome variable. Conclusions/ interpretation: These findings are the first to suggest that oral vitamin C supplementation provides an effective prophylaxis against exercise-induced free radical-mediated lipid peroxidation in human diabetic blood.

The Effects of Cilostazol on Exercise-induced Ischaemia-reperfusion Injury in Patients with Peripheral Arterial Disease

Objectives: Cilostazol improves walking distance in peripheral arterial disease (PAD) patients. The study objectives were to assess the effects of cilostazol on walking distance, followed by the additional assessment of cilostazol on exercise-induced ischaemiaereperfusion injury in such patients.

Methods: PAD patients were prospectively recruited to a double-blinded, placebo-controlled trial. Patients were randomised to receive either cilostazol 100 mg or placebo twice a day. The primary end-point was an improvement in walking distance. Secondary end-points included the assessment of oxygen-derived free-radical generation, antioxidant consumption and other markers of the in?ammatory cascade. Initial and absolute claudication distances (ICDs and ACDs, respectively) were measured on a treadmill. In?ammatory response was assessed before and 30 min post-exercise by measuring lipid hydroperoxide, ascorbate, atocopherol, b-carotene, P-selectin, intracellular and vascular cell-adhesion molecules (I-CAM and V-CAM), thromboxane B2 (TXB2), interleukin-6, interleukin-10, high-sensitive C-reactive protein (hsCRP), albuminecreatinine ratio (ACR) and urinary levels of p75TNF receptor. All tests were performed at baseline and 6 and 24 weeks.

Results: One hundred and six PAD patients (of whom 73 were males) were recruited and successfully randomised from December 2004 to January 2006. Patients who received cilostazol demonstrated a more signi?cant improvement in the mean percentage change from baseline in ACD (77.2% vs. 26.6% at 6 weeks, pZ0.026 and 161.7% vs. 79.0% at 24 weeks, pZ0.048) as compared to the placebo. Cilostazol reduced lipid hydroperoxide levels compared to a placebo-related increase before and after exercise (6 weeks: pre-exercise: 11.8% vs.

Influence of the anion on the electrodeposition of cobalt from imidazolium ionic liquids

Cyclic voltammetry and absorption spectrophotometry were used to examine the complex formation of cobalt (II) in the ionic liquids 1-butyl-3-methylimidazolium chloride ([C(4)mim] Cl) and 1-butyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide ([C(4)mim][Tf2N]). In [C(4)mim]Cl, cobalt(II) is complexed as [CoCl4](2-) at CoCl2 concentrations less than 33 mol %. Cyclic voltammograms show that cobalt cannot be electrodeposited at these concentrations. However, cobalt metal can be electrodeposited at CoCl2 concentrations above the threshold concentration of 33 mol %. In the ionic liquid [C(4)mim][Tf2N] there is no threshold CoCl2 concentration for electrodeposition due to the absence of [CoCl4](2-). (C) 2007 The Electrochemical Society.

Sedentary aging increases resting and exercise-induced intramuscular free radical formation

Mitochondrial free radical formation has been implicated as a potential mechanism underlying degenerative senescence, although human data are lacking. Therefore, the present study was designed to examine if resting and exercise-induced intramuscular free radical-mediated lipid peroxidation is indeed increased across the spectrum of sedentary aging. Biopsies were obtained from the vastus lateralis in six young (26 ± 6 yr) and six aged (71 ± 6 yr) sedentary males at rest and after maximal knee extensor exercise. Aged tissue exhibited greater (P < 0.05 vs. the young group) electron paramagnetic resonance signal intensity of the mitochondrial ubisemiquinone radical both at rest (+138 ± 62%) and during exercise (+143 ± 40%), and this was further complemented by a greater increase in a-phenyl-tert-butylnitrone adducts identified as a combination of lipid-derived alkoxyl-alkyl radicals (+295 ± 96% and +298 ± 120%). Lipid hydroperoxides were also elevated at rest (0.190 ± 0.169 vs. 0.148 ± 0.071 nmol/mg total protein) and during exercise (0.567 ± 0.259 vs. 0.320 ± 0.263 nmol/mg total protein) despite a more marked depletion of ascorbate and uptake of a/ß-carotene, retinol, and lycopene (P < 0.05 vs. the young group). The impact of senescence was especially apparent when oxidative stress biomarkers were expressed relative to the age-related decline in mitochondrial volume density and absolute power output at maximal exercise. In conclusion, these findings confirm that intramuscular free radical-mediated lipid peroxidation is elevated at rest and during acute exercise in aged humans.

Asymmetric synthesis of an N-acylpyrrolidine for inhibition of HCV polymerase

A practical asymmetric synthesis of a highly substituted N-acylpyrrolidine on multi-kilogram scale is described. The key step in the construction of the three stereocenters is a [3+2] cycloaddition of methyl acrylate and an imino ester prepared from L-leucine t-butyl ester hydrochloride and 2-thiazolecarboxaldehyde. The cycloaddition features novel asymmetric catalysis via a complex of silver acetate and a cinchona alkaloid, particularly hydroquinine, with complete diastereomeric control and up to 87% enantiomeric control. The alkaloid serves as a ligand as well as a base for the formation of the azomethine ylide or 1,3-dipole. Experiments have shown that the hydroxyl group of hydroquinine is a critical element for the enantioselectivities observed. The cycloaddition methodology is also applicable to methylvinyl ketone, providing access to either alpha- or beta-epimers of 4-acetylpyrrolidine depending on the reaction conditions utilized. The synthesis also highlights an efficient N-acylation, selective O- versus N-methylation, and a unique ester reduction with NaBH4-MeOH catalyzed by NaB(OAc)(3)H that not only achieves excellent chemoselectivity but also avoids formation of the undesired but thermodynamically favored epimer. The highly functionalized target is synthesized in seven linear steps from L-leucine t-butyl ester hydrochloride with all three isolated intermediates being highly crystalline.

Synthesis of the alkaloids hopromine, hoprominol and hopromalinol, using transamidation methods.

Synthesis of the unsym. Homalium alkaloids hopromine (I, R = H, R1 = pentyl), hoprominol (I, R = OH, R1 = pentyl) and hopramalinol (I, R = OH, R1 = Ph), in diastereoisomeric mixt. form, is reported. The component eight-membered azalactams are first prepd. N-(3-halogenopropyl)-4-pentyl- and 4-heptylazetidin-2-ones are aminated and ring expanded in liq. ammonia to give, after reductive methylation, the corresponding 4-alkyl-5-methyl-1,5-diazacyclooctan-2-ones. Synthesis of the 4-(2-hydroxyheptyl)-5-methyl-1,5-diazacyclooctan-2-one required for hoprominol and hopromalinol is carried out via 4-allyl ?-lactam ring expansion to the eight-membered 4-allylazalactam, followed by methylation, epoxidn. and epoxide opening with lithium dibutylcuprate. A similar epoxidn.-cuprate sequence was carried out on the epoxypropyl ?-lactam, as its N-tert-butyldimethylsilyl deriv., and led to a convenient copper-catalyzed N- to O-migration of the protection; this migration is examd. Alkylation gave O-tert-butyldimethylsilyl-protected N-(3-chloropropyl)-4-(2-hydroxyheptyl)azetidin-2-one which could be aminated and transamidated in excellent yield, to give, after methylation, a superior sequence to the required eight-membered hydroxy azalactam. Although satisfactory for attachment of the first azalactam unit, a dibromobutane coupling system proved unreactive for the second. Couplings with unmethylated, methylated, and benzyloxycabronyl-protected azalactams were examd. using (E)-1,4-dibromobutene and (Z)-1,4-dichlorobutene as the bridging unit. Employing the latter, coupling the first N-methylated azalactam with potassium bis(trimethylsilyl)amide as the base, and then the second with bis(trimethylsilyl)amide-sodium hydride as the base system, provided a satisfactory synthetic outcome. Hydrogenation under acidic conditions gave the unsym. structures hopromine, hoprominol and hopromalinol, as well as the more simple and sym. alkaloid, homaline.

Small angle neutron scattering from 1-alkyl-3-methylimidazolium hexafluorophosphate ionic liquids ([C(n)mim][PF6], n=4, 6, and 8)

The presence of local anisotropy in the bulk, isotropic, and ionic liquid phases-leading to local mesoscopic inhomogeneity-with nanoscale segregation and expanding nonpolar domains on increasing the length of the cation alkyl-substituents has been proposed on the basis of molecular dynamics (MD) simulations. However, there has been little conclusive experimental evidence for the existence of intermediate mesoscopic structure between the first/second shell correlations shown by neutron scattering on short chain length based materials and the mesophase structure of the long chain length ionic liquid crystals. Herein, small angle neutron scattering measurements have been performed on selectively H/D-isotopically substituted 1-alkyl-3-methylimidazolium hexafluorophosphate ionic liquids with butyl, hexyl, and octyl substituents. The data show the unambiguous existence of a diffraction peak in the low-Q region for all three liquids which moves to longer distances (lower Q), sharpens, and increases in intensity with increasing length of the alkyl substituent. It is notable, however, that this peak occurs at lower values of Q (longer length scale) than predicted in any of the previously published MD simulations of ionic liquids, and that the magnitude of the scattering from this peak is comparable with that from the remainder of the amorphous ionic liquid. This strongly suggests that the peak arises from the second coordination shells of the ions along the vector of alkyl-chain substituents as a consequence of increasing the anisotropy of the cation, and that there is little or no long-range correlated nanostructure in these ionic liquids.

Interfacial tensions of imidazolium-based ionic liquids with water and n-alkanes

The interfacial tension of the liquid-liquid phase boundary of several 1,3-dialkyl imidazolium based ionic liquids, namely, 1,3-dimethylimidazolium bis{(trifluoromethyl)sulfonyl}imide [C(1)mim][NTf2], 1-ethyl-3-methylimidazoliurn bis{(trifluoromethyl)sulfonyl}imide [C(2)mim][NTf2], 1-butyl-3-methylimidazolium bis{(trifluoromethyl)sulfonyl}imide [C(4)mim][NTf2], 1-hexyl-3-methylimidazolium bis{(trifluoromethyl)sulfonyl}imide [C(6)mim][NTf2], 1-octyl-3-methylimidazolium bis{(trifluoromethyl)sulfonyl}imide [C(8)mim][NTf2], 1-butyl-3-methylimidazolium trifluoromethylsulfonate [C(4)mim][CF3SO3], and 1-butyl-3-methylimidazolium trifluoroacetate [C(4)mim][CF3COO] with water and with the n-alkanes, n-hexane, n-octane and n-decane, has been measured using the pendant drop method as a function of temperature from 293 to 323 K. The experimental interfacial tension data were correlated using the ionic liquid parachor estimation method and a mutual solubility model. The influence of the cation and anion of ionic liquids and also of alkyl chain length of n-alkanes on interfacial tension is discussed. It has also been demonstrated that the interfacial tension data estimated by the correlation methods are in good agreement with the experimental data. (C) 2010 Elsevier B.V. All rights reserved.

Selecting Room-Temperature Ionic Liquids to Optimize Voltammetric Responses: The Oxidation of NADH

The electrochemistry of nicotinamide adenine dinucleotide (NADH) in its reduced form was examined in two room-temperature ionic liquids (RTILs): 1-ethyl-3-methylimidazolium bis-(trifluoromethylsulfonyl)imide ([C(2)mim][NTf2]) and 1-butyl-3-methylimidazolium hexafluorophos-phate ([C(4)mim][PF6]). NADH oxidation has previously been studied in aqueous solution where it follows the pathway: one-electron oxidation to the NADH(center dot+) radical cation, deprotonation to produce the neutral NAD(center dot) radical, then oxidation to the NAD(+) cation. The electrochemistry of NADH was examined in [C(2)mim][NTf2] and [C(4)mim][PF6] at the bare Pt electrode (10 mu m diameter): In [C(2)mim][NTf2], no oxidation was observed; in [C(4)mim][PF6], an oxidative signal was observed, which likely followed the pathway described above, where upon formation of the NADH(center dot+) radical cation, the [PF6](-) anion (unlike the [NTf2](-) anion) reacts with the proton to form HPF6, which decomposes. This demonstrates the tunability of RTILs, whereby the choice of one anion in an RTIL over another can promote a reaction. Poly(vinylferrocene) (PVF) was studied as a mediator for the NADH detection in both RTILs to attempt to lower the potential of NADH detection. The Pt electrode was modified with PVF, and the oxidation of PVF to PVF+ was observed in [C(2)mim][NTf2] and [C(4)mim][PF6], but no mediation of the NADH oxidation was observed.

The electrochemical reduction of the purines guanine and adenine at platinum electrodes in several room temperature ionic liquids

The reduction of guanine was studied by microelectrode voltammetry in the room temperature ionic liquids (RTILs) N-hexyltriethylammonium his (trifluoromethanesulfonyl) imide [N-6.2.2.2][N(Tf)(2)], 1-butyl-3-methylimidazolium hexafluorosphosphate [C(4)mim] [PF6], N-butyl-N-methyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide [C(4)mpyrr][N(Tf)(2)], 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C-4mim][N(TF)(2)], N-butyl-N-methyl-pyrrolidinium dicyanamide [C(4)mpyrr][N(NC)(2)] and tris(P-hexyl)-tetradecylphosphonium trifluorotris(pentafluoroethyl)phosphate [P-14,P-6,(6,6)][FAP] on a platinum microelectrode. In [N-6,N-2,N-2,N-2][NTf2] and [P-14,P-6,P-6.6][FAP], but not in the other ionic liquids studied, guanine reduction involves a one-electron, diffusion-controlled process at very negative potential to produce an unstable radical anion. which is thought to undergo a dimerization reaction, probably after proton abstraction from the cation of the ionic liquid. The rate of this subsequent reaction depends on the nature of the ionic liquid, and it is faster in the ionic liquid [P-14,P-6,P-6.6[FAP], in which the formation of the resulting dimer can be voltammetrically monitored at less negative potentials than required for the reduction of the parent molecule. Adenine showed similar behaviour to guanine but the pyrimidines thymine and cytosine did not; thymine was not reduced at potentials less negative than required for solvent (RTIL) decomposition while only a poorly defined wave was seen for cytosine. The possibility for proton abstraction from the cation in [N-6,N-2,N-2,N-2],[NTF2] and [P-14,P-6,P-6.6][FAP] is noted and this is thought to aid the electrochemical dimerization process. The resulting rapid reaction is thought to shift the reduction potentials for guanine and adenine to lower values than observed in RTILs where the scope for proton abstraction is not present. Such shifts are characteristic of so-called EC processes where reversible electron transfer is followed by a chemical reaction. (C) 2009 Elsevier B.V. All rights reserved.

ZnO nanofluids: Green synthesis, characterization, and antibacterial activity

Zinc oxide nanoparticles have been synthesized by microwave decomposition of zinc acetate precursor using an ionic liquid, 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imide, [bmim][NTf2] as a green solvent. The structure and morphology of ZnO nanoparticles have been characterized using X-ray diffraction and transmission electron microscopy. The ZnO nanofluids have been prepared by dispersing ZnO nanoparticles in glycerol as a base fluid in the presence of ammonium citrate as a dispersant. The antibacterial activity of suspensions of ZnO nanofluids against (E. coli) has been evaluated by estimating the reduction ratio of the bacteria treated with ZnO. Survival ratio of bacteria decreases with increasing the concentrations of ZnO nanofluids and time. The results show that an increase in the concentrations of ZnO nanofluids produces strong antibacterial activity toward E. coli. (C) 2010 Elsevier B.V. All rights reserved.

Tissue biomarker development in a multicentre trial context: a feasibility study on the PETACC3 stage II and III colon cancer adjuvant treatment trial.

Purpose: We evaluated the feasibility of biomarker development in the context of multicenter clinical trials.

Experimental Design: Formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from a prospective adjuvant colon cancer trial (PETACC3). DNA was isolated from tumor as well as normal tissue and used for analysis of microsatellite instability, KRAS and BRAF genotyping, UGT1A1 genotyping, and loss of heterozygosity of 18 q loci. Immunohistochemistry was used to test expression of TERT, SMAD4, p53, and TYMS. Messenger RNA was retrieved and tested for use in expression profiling experiments.

Results: Of the 3,278 patients entered in the study, FFPE blocks were obtained from 1,564 patients coming from 368 different centers in 31 countries. In over 95% of the samples, genomic DNA tests yielded a reliable result. Of the immmunohistochemical tests, p53 and SMAD4 staining did best with reliable results in over 85% of the cases. TERT was the most problematic test with 46% of failures, mostly due to insufficient tissue processing quality. Good quality mRNA was obtained, usable in expression profiling experiments.

Conclusions: Prospective clinical trials can be used as framework for biomarker development using routinely processed FFPE tissues. Our results support the notion that as a rule, translational studies based on FFPE should be included in prospective clinical trials.

Application Of the Michaelis-Arbuzov Reaction to the Synthesis Of Internucleoside 3'-S-Phosphorothiolate Linkages

The 5'-O-monomethoxytrityl-3'-S-(aryldisulfanyl)-3'-deoxythymidines 7 and 8 have been prepared by the reaction of 5'-O-monomethoxytrityl-3'-thiothymidine with the appropriate arenesulfenyl chloride. These disulfides undergo a Michaelis–Arbusov reaction with simple trialkyl phosphites to yield 5'-O-monomethoxytrityl-3'-thiothymidin-3'-yl O,O-dialkyl phosphorothiolates. More interestingly, 3'-deoxy-3'-S-(2, 4-dinitrophenylsulfanyl)-5'-O-monomethoxytritylthymidine 8 reacts with a variety of thymidin-5'-yl dialkyl phosphites to give dithymidine phosphorothiolate triesters with the phosphorothiolate group protected with either a methyl or a 2-cyanoethyl group. 3'-O-(tert-Butyldimethylsilyl)thymidin-5'-yl triethylammoniumphosphonate 17 is converted into the corresponding bis-(O-trimethylsilyl) phosphite by treatment with bis(trimethylsilyl)trifluoroacetamide. in situ Reaction of this phosphate with disulfide 8 gives, after work-up, the dithymidine phosphorothiolate diester directly. Methylation of compound 17 with methyl chloromethanoate, followed by silylation and subsequent reaction with disulfide 8, gives the methyl-protected dithymidine phosphorothiolate triester.

Effect of activated carbon xerogel pore size on the capacitance performance of ionic liquid electrolytes

The use of ionic liquid (IL) electrolytes promises to improve the energy density of electrochemical capacitors (ECs) by allowing for operation at higher voltages. Several studies have also shown that the pore size distribution of materials used to produce electrodes is an important factor in determining EC performance. In this research the capacitative, energy and power performance of ILs 1-ethyl-3- methylimidazolium tetrafluoroborate (EMImBF4), 1-ethyl-3-methylimidazolium dicyanamide (EMImN(CN)2), 1,2-dimethyl-3-propylimidazolium bis(trifluoromethylsulfonyl)imide (DMPImTFSI), and 1-butyl-3-methylpyrrolidinium tris(pentafluoroethyl)trifluorophosphate (BMPyT(F5Et)PF3) were studied and compared with the commercially utilised organic electrolyte 1M tetraethylammonium tetrafluoroborate solution in anhydrous propylene carbonate (Et4NBF4–PC 1 M). To assess the effect of pore size on IL performance, controlled porosity carbons were produced from phenolic resins activated in CO2. The carbon samples were characterised by nitrogen adsorption– desorption at 77 K and the relevant electrochemical behaviour was characterised by cyclic voltammetry, galvanostatic charge–discharge and electrochemical impedance spectroscopy. The best capacitance performance was obtained for the activated carbon xerogel with average pore diameter 3.5 nm, whereas the optimum rate performance was obtained for the activated carbon xerogel with average pore diameter 6 nm. When combined in an EC with IL electrolyte EMImBF4 a specific capacitance of 210 F g1 was obtained for activated carbon sample with average pore diameter 3.5 nm at an operating voltage of 3 V. The activated carbon sample with average pore diameter 6 nm allowed for maximum capacitance retention of approximately 70% at 64 mA cm2.

Short Isoforms of the Cold Receptor TRPM8 Inhibit Channel Gating by Mimicking Heat Action Rather than Chemical Inhibitors

Transient receptor potential (TRP) channels couple various environmental factors to changes in membrane potential, calcium influx, and cell signaling. They also integrate multiple stimuli through their typically polymodal activation. Thus, although the TRPM8 channel has been extensively investigated as the major neuronal cold sensor, it is also regulated by various chemicals, as well as by several short channel isoforms. Mechanistic understanding of such complex regulation is facilitated by quantitative single-channel analysis. We have recently proposed a single-channel mechanism of TRPM8 regulation by voltage and temperature. Using this gating mechanism, we now investigate TRPM8 inhibition in cell-attached patches using HEK293 cells expressing TRPM8 alone or coexpressed with its short sM8-6 isoform. This is compared with inhibition by the chemicals N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC) and clotrimazole or by elevated temperature. We found that within the seven-state single-channel gating mechanism, inhibition of TRPM8 by short sM8-6 isoforms closely resembles inhibition by increased temperature. In contrast, inhibition by BCTC and that by clotrimazole share a different set of common features. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.