High-Pressure Low-Temperature Locknut Cell For Both Electron-Paramagnetic-Res And Nmr-Studies To 10 Kilobars And 77-K

A locked high-pressure cell with working pressure range up to 10 kbars suitable for low-temperature studies to 77 K has been described. It can be used for both EPR and NMR studies of single crystals (and other solid samples). The high-pressure seal and all other aspects of the cell remain the same for either application. Only a change of the bottom plug is required for a switch from a nuclear-magnetic-resonance (NMR) to an electron-paramagnetic-resonance (EPR) experiment. Details of the procedure for the calibration of pressure inside the cell at various temperatures are discussed. The performance of the cell in EPR (Cr3+ion) and NMR (27Al nucleus) studies is reported.

Quadratic tranverse anisotropy term due to dislocations in Mn12 acetate crystals directly observed by EPR spectroscopy

High-sensitivity electron paramagnetic resonance experiments have been carried out in fresh and stressed Mn12 acetate single crystals for frequencies ranging from 40 GHz up to 110 GHz. The high number of crystal dislocations formed in the stressing process introduces a E(Sx2-Sy2) transverse anisotropy term in the spin Hamiltonian. From the behavior of the resonant absorptions on the applied transverse magnetic field we have obtained an average value for E=22 mK, corresponding to a concentration of dislocations per unit cell of c=10-3.

Two-phase separation observed in thermally treated pellets of ClO4- doped poly(3-methylthiophene) from electron spin resonance measurements

A pressed pellet of CIO (-)(4) poly (3-methylthiophene) (P3MT) was heated for two hours at 85 degrees C and suddenly dropped in liquid nitrogen. A change was observed around 220 K in the Electron Spin Resonance (ESR) spectra when the sample was slowly cooled from room temperature. ESR line asymmetry parameter (A/B) showed two spatially separated phases. One was identified as a small metallic-like phase. The other phase, the larger one, makes a transition to a semiconducting Charge-Density Wave (CDW) state.

Assessing the functional structure of molecular transporters by EPR spectroscopy

In this thesis, the self-assembled functional structure of a broad range of amphiphilic molecular transporters is studied. By employing paramagnetic probe molecules and ions, continuous-wave and pulse electron paramagnetic resonance spectroscopy reveal information about the local structure of these materials from the perspective of incorporated guest molecules. First, the transport function of human serum albumin for fatty acids is in the focus. As suggested by the crystal structure, the anchor points for the fatty acids are distributed asymmetrically in the protein. In contrast to the crystallographic findings, a remarkably symmetric entry point distribution of the fatty acid binding channels is found, which may facilitate the uptake and release of the guest molecules. Further, the metal binding of 1,2,3-triazole modified star-shaped cholic acid oligomers is studied. These biomimetic molecules are able to include and transport molecules in solvents of different polarity. A pre-arrangement of the triazole groups induces a strong chelate-like binding and close contact between guest molecule and metal ion. In absence of a preordering, each triazole moiety acts as a single entity and the binding affinity for metal ions is strongly decreased. Hydrogels based on N-isopropylacrylamide phase separate from water above a certain temperature. The macroscopic thermal collapse of these hydrogels is utilized as a tool for dynamic nuclear polarization. It is shown that a radical-free hyperpolarized solution can be achieved with a spin-labeled gel as separable matrix. On the nanoscale, these hydrogels form static heterogeneities in both structure and function. Collapsed regions protect the spin probes from a chemical decay while open, water-swollen regions act as catalytic centers. Similarly, thermoresponsive dendronized polymers form structural heterogeneities, which are, however, highly dynamic. At the critical temperature, they trigger the aggregation of the polymer into mesoglobules. The dehydration of these aggregates is a molecularly controlled non-equilibrium process that is facilitated by a hydrophobic dendritic core. Further, a slow heating rate results in a kinetically entrapped non-equilibrium state due to the formation of an impermeable dense polymeric layer at the periphery of the mesoglobule.

Synthesis and properties of fullerene C60 and C70 spin probes containing isoindoline nitroxides

Three fullerene isoindoline nitroxides N-methyl-3,4-fulleropyrrolidine-2-spiro-5′- (1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl), (C60-(TMIO)m, and C70-(TMIO)n) were synthesized by the covalent bonding of 5-formyl-1,1,3,3-tetramethyl isoindolin-2-yloxyl to the fullerenes C60 and C70. Significantly, the X-ray photoelectron spectra indicated the characteristic N 1s signals of NO. at 402 eV. The atomic force microscope morphologies showed that the average particle sizes of C60-(TMIO)m and C70-(TMIO)n were 38 and 15 nm. The electrochemical experiments indicated that fullerene bound isoindoline nitroxides retained similar electrochemical properties and redox reaction mechanisms as the parent nitroxides. The electron paramagnetic resonance spectra of the fullerene isoindoline nitroxides all exhibited the hyperfine splittings and characteristic spectra of tetramethyl isoindoline nitroxides, with typical nitroxide g-values and nitrogen isotropic hyperfine coupling constants. Therefore, these fullerene isoindoline nitroxides may be considered as potential candidates for novel biological spin probes using electron paramagnetic resonance spectroscopy.

Synthesis of some copper(II)-chelating (dialkylamino)pyridine amphiphiles and evaluation of their esterolytic capacities in cationic micellar media

Three new (dialkylamino)pyridine (DAAP)-based ligand amphiphiles 3-5 have been synthesized. All of the compounds possess a metal ion binding subunit in the form of a 2,6-disubstituted DAAP moiety. In addition, at least one ortho-CH2OH substituent is present in all the ligands. Complex formation by these ligands with various metal ions were examined under micellar conditions, but only complexes with Cu(II) ions showed kinetically potent esterolytic capacities under micellar conditions. Complexes with Cu(II) were prepared in host comicellar cetyltrimethylammonium bromide (CTABr) media at pH 7.6. Individual complexes were characterized by UV-visible absorption spectroscopy and electron paramagnetic resonance spectroscopy. These metallomicelles speed the cleavage of the substrates p-nitrophenyl hexanoate or p-nitrophenyl diphenyl phosphate. To ascertain the nature of the active esterolytic species, the stoichiometries of the respective Cu(II) complexes were determined from the kinetic version of Job's plot. In all the instances, 2:1 complex ligand/Cu(II) ion are the most kinetically competent species. The apparent pK(a) values of the Cu(II)-coordinated hydroxyl groups of the ligands 3, 4, and 5, in the comicellar aggregate, are 7.8, 8.0, and 8.0, respectively, as estimated from the rate constant vs pH: profiles of the ester cleavage reactions. The nucleophilic metallomicellar reagents and the second-order "catalytic" rate constants toward esterolysis of the substrate p-nitrophenyl hexanoate (at 25 degrees C, pH 7.6) are 37.5 for 3, 11.4 for 4, and 13.8 for 5. All catalytic systems comprising the coaggregates of 3, 4, or 5 and CTABr demonstrate turnover behavior in the presence of excess substrate.

A new perovskite series based on lanthanum chromite [LaCr1−xMxO3−δ]

A new series of substituted perovskites of the type LaCr1−xMxO3−δ, where M=Cu or Mg have been synthesised by the citrate gel process and characterized by means of powder X-ray diffraction, infrared spectroscopy, selected area diffraction and also by electron paramagnetic resonance spectroscopy. The general powder morphology was also observed using scanning electron microscopy. 40 mole percent substitution of Cr3+ by Cu2+ or Mg2+ have shown to result in single phase perovskite structure. Beyond x=0.5, a new phase has been identified in a narrow compositional range. Effect of Cu and Mg substitution on the sinterability of pure LaCrO3 has also been studied. It is possible to get near theoretically dense materials at a temperature as low as 1200°C in air by copper substitution.

Studies on Cu/CeO2: A new NO reduction catalyst

Fine particle and large surface area Cu/CeO2 catalysts of crystallite sizes in the range of 100-200 Angstrom synthesized by the solution combustion method have been investigated for NO reduction. Five percent Cu/CeO2 catalyst shows nearly 100% conversion of NO by NH3 below 300 degrees C, whereas pure ceria and Zr, Y, and Ca doped ceria show 85-95% NO conversion above 600 degrees C. Similarly NO reduction by CO has been observed over 5% Cu/CeO2 with nearly 100% conversion below 300 degrees C. Hydrocarbon (n-butane) oxidation by NO to CO2, N-2, and H2O has also been demonstrated over this catalyst below 350 degrees C making Cu/CeO2 a new NO reduction catalyst in the low temperature window of 150-350 degrees C. Kinetics of NO reduction over 5% Cu/CeO2 have also been investigated. The rate constants are in the range of 1.4 x 10(4) to 2.3 x 10(4) cm(3) g(-1) s(-1) between 170 and 300 degrees C. Cu/CeO2 catalysts are characterized by X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy, and electron paramagnetic resonance spectroscopy where Cu2+ ions are shown to be dispersed on the CeO2 surface. (C) 1999 Academic Press.

Graphene scavenges free radicals to synergistically enhance structural properties in a gamma-irradiated polyethylene composite through enhanced interfacial interactions

A unique strategy for scavenging free radicals in situ on exposure to gamma irradiation in polyethylene (PE) nanocomposites is presented. Blends of ultra-high molecular weight PE and linear low-density PE (PEB) and their nanocomposites with graphene (GPEB) were prepared by melt mixing to develop materials for biomedical implants. The effect of gamma irradiation on the microstructure and mechanical properties was systematically investigated. The neat blend and the nanocomposite were subjected to gamma-ray irradiation in order to improve the interfacial adhesion between PE and graphene sheets. Structural and thermal characterization revealed that irradiation induced crosslinking and increased the crystallinity of the polymer blend. The presence of graphene further enhanced the crystallinity via crosslinks between the polymer matrix and the filler on irradiation. Graphene was found to scavenge free radicals as confirmed by electron paramagnetic resonance spectroscopy. Irradiation of graphene-containing polymer composites resulted in the largest increase in modulus and hardness compared to either irradiation or addition of graphene to PEB alone. This study provides new insight into the role of graphene in polymer matrices during irradiation and suggests that irradiated graphene-polymer composites could emerge as promising materials for use as articulating surfaces in biomedical implants.

DNA-mediated charge transport signaling within the cell

DNA possesses the curious ability to conduct charge longitudinally through the π-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.

Herein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.

In separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?

UvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.

Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness?

Bailey DM, Taudorf S, Berg RMG, Lundby C, McEneny J, Young IS, Evans KA, James PE, Shore A, Hullin DA, McCord JM, Pedersen BK, Moller K. Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness? Am J Physiol Regul Integr Comp Physiol 297: R1283-R1292, 2009. First published September 2, 2009; doi: 10.1152/ajpregu.00366.2009.-This study examined whether hypoxia causes free radical-mediated disruption of the blood-brain barrier (BBB) and impaired cerebral oxidative metabolism and whether this has any bearing on neurological symptoms ascribed to acute mountain sickness (AMS). Ten men provided internal jugular vein and radial artery blood samples during normoxia and 9-h passive exposure to hypoxia (12.9% O-2). Cerebral blood flow was determined by the Kety-Schmidt technique with net exchange calculated by the Fick principle. AMS and headache were determined with clinically validated questionnaires. Electron paramagnetic resonance spectroscopy and ozone-based chemiluminescence were employed for direct detection of spin-trapped free radicals and nitric oxide metabolites. Neuron-specific enolase (NSE), S100 beta, and 3-nitrotyrosine (3-NT) were determined by ELISA. Hypoxia increased the arterio-jugular venous concentration difference (a-v(D)) and net cerebral output of lipid-derived alkoxyl-alkyl free radicals and lipid hydroperoxides (P

Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia

ABSTRACT BACKGROUND: Acute exposure to high-altitude stimulates free radical formation in lowlanders yet whether this persists during chronic exposure in healthy well-adapted and maladapted highlanders suffering from chronic mountain sickness (CMS) remains to be established. METHODS: Oxidative-nitrosative stress [ascorbate radical (A•-), electron paramagnetic resonance spectroscopy and nitrite (NO2-), ozone-based chemiluminescence] was assessed in venous blood of 25 male highlanders living at 3,600 m with (n = 13, CMS+) and without (n = 12, CMS-) CMS. Twelve age and activity-matched healthy male lowlanders were examined at sea-level and during acute hypoxia. We also measured flow-mediated dilatation (FMD), arterial stiffness (AIx-75) and carotid intima-media thickness (IMT). RESULTS: Compared to normoxic lowlanders, oxidative-nitrosative stress was moderately increased in CMS- (P < 0.05) as indicated by elevated A•- (3,191 ± 457 vs. 2,640 ± 445 arbitrary units (AU)] and lower NO2- (206 ± 55 vs. 420 ± 128 nmol/L) whereas vascular function remained preserved. This was comparable to that observed during acute hypoxia in lowlanders in whom vascular dysfunction is typically observed. In contrast, this response was markedly exaggerated in CMS+ (A•-: 3,765 ± 429 AU and NO2- : 148 ± 50 nmol/L) compared to both CMS- and lowlanders (P < 0.05). This was associated with systemic vascular dysfunction as indicated by lower (P < 0.05 vs. CMS-) FMD (4.2 ± 0.7 vs. 7.6 ± 1.7 %) and increased AIx-75 (23 ± 8 vs. 12 ± 7 %) and carotid IMT (714 ± 127 vs. 588 ± 94 µM). CONCLUSIONS: Healthy highlanders display a moderate sustained elevation in oxidative-nitrosative stress that unlike the equivalent increase evoked by acute hypoxia in healthy lowlanders, failed to affect vascular function. Its more marked elevation in patients with CMS may contribute to systemic vascular dysfunction.Clinical Trials Gov Registration # NCT011827921Neurovascular Research Laboratory, Faculty of Health, Science and Sport, University of Glamorgan, Wales, UK;2Sondes Moléculaires en Biologie et Stress Oxydant, Institut de Chimie Radicalaire, CNRS UMR 7273, Aix-Marseille University, France;3Department of Cardiology, University Hospital of Bern, Bern, Switzerland;4Institute of Clinical Physiology, CNR, Pisa, Italy;5Instituto Bolivano de Biologia de Altura, La Paz, Bolivia;6Centre for Clinical and Population Sciences, Queen's University Belfast, Belfast, Northern Ireland,7Botnar Center for Clinical Research, Hirslanden Group, Lausanne, Switzerland;8Facultad de Ciencias, Departamento de Biología, Universidad de Tarapacá, Arica, Chile and9Department of Internal Medicine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland*Drs Bailey, Rimoldi, Scherrer and Sartori contributed equally to this workCorrespondence: Damian Miles Bailey, Neurovascular Research Laboratory, Faculty of Health, Science and Sport, University of Glamorgan, UK CF37 4AT email: dbailey1@glam.ac.uk.