Purely Spatial Quantum Diffusion of H Atoms in Solid H_{2} at Temperatures below 1 K.

We report on a direct measurement of the quantum diffusion of H atoms in solid molecular hydrogen films at T=0.7 K. We obtained a rate of pure spatial diffusion of H atoms in the H_{2} films, D^{d}=5(2)×10^{-17} cm^{2} s^{-1}, which was 2 orders of magnitude faster than that obtained from H atom recombination, the quantity used in all previous work to characterize the mobility of H atoms in solid H_{2}. We also observed that the H-atom diffusion was significantly enhanced by injection of phonons. Our results provide the first measurement of the pure spatial diffusion rate for H atoms in solid H_{2}, the only solid state system beside ^{3}He-^{4}He mixtures, where atomic diffusion does not vanish even at temperatures below 1 K.

Dynamic nuclear polarization and ESR hole burning in As doped silicon.

We present an experimental study of the Dynamic Nuclear Polarization (DNP) of 29Si nuclei in silicon crystals of natural abundance doped with As in the temperature range 0.1-1 K and in a strong magnetic field of 4.6 T. This ensures a very high degree of electron spin polarization, extremely slow nuclear relaxation and optimal conditions for realization of Overhauser and resolved solid effects. We found that the solid effect DNP leads to the appearance of a pattern of holes and peaks in the ESR line, separated by the super-hyperfine interaction between the donor electron and 29Si nuclei closest to the donor. On the contrary, the Overhauser effect DNP mainly affects the remote 29Si nuclei having the weakest interaction with the donor electron. This leads to the appearance of a very narrow (≈3 mG wide) hole in the ESR line. We studied relaxation of the holes after burning, which is caused by the nuclear spin diffusion. Analyzing the dynamics of the hole in the spectrum with a simple one-dimensional diffusion model leads to a value of the diffusion coefficient D = 8(3) × 10-9 G2 s-1. Our data indicate that the spin diffusion is not completely prevented even in the frozen core near the donors. The emergence of the narrow hole after the Overhauser DNP may be explained by a partial "softening" of the frozen core caused by decoupling of the donor electron and remote 29Si nuclei.

Formation of Nuclear-Polarized Phases of H Atoms Embedded in Solid H_{2} Films.

We report on an experimental observation of two phases of hydrogen atoms in solid H_{2} films at temperatures of 0.1-0.8 K, characterized by a large enhancement of the nuclear spin polarization compared to that given by Boltzmann statistics (p=0.15 at T=0.15 K). The first phase with p=0.35(5) is formed spontaneously during sample storage in a high magnetic field (B=4.6 T). The second phase with an even higher nuclear polarization, p=0.75(7), can be achieved at T≤0.55 K by repeating sequences of dynamic nuclear polarization followed by a system relaxation. Upon warming through the range 0.55-0.65 K, the highly nuclear-polarized phase undergoes a phase transition to the spontaneously polarized phase which breaks down at T≃0.8 K, and the nuclear polarization gradually converges to the Boltzmann distribution. We discuss possible scenarios for explaining the nature of the observed phenomena.

ESR study of atomic hydrogen and tritium in solid T2 and T2:H2 matrices below 1 K.

We report on the first ESR study of atomic hydrogen and tritium stabilized in solid T2 and T2:H2 matrices down to 70 mK. The concentrations of T atoms in pure T2 approached 2 × 1020 cm-3 (0.60%) and record-high concentrations of H atoms ∼1 × 1020 cm-3 (0.33%) were reached in T2:H2 solid mixtures where a fraction of T atoms became converted into H due to the isotopic exchange reaction T + H2 → TH + H. The maximum concentrations of unpaired T and H atoms were limited by their recombination which becomes enhanced by efficient atomic diffusion due to the presence of a large number of vacancies and phonons generated in the matrices by ß-particles. Recombination also appeared in an explosive manner, both being stimulated and spontaneously in thick films where sample cooling was insufficient. We suggest that the main mechanism for H and T migration is physical diffusion related to tunneling or hopping to vacant sites in contrast to tunneling chemical exchange reactions which govern diffusion of H and D atoms created in H2 and D2 matrices by other methods.

Tunneling chemical exchange reaction D + HD → D2 + H in solid HD and D2 at temperatures below 1 K.

We report on a study of the exchange tunneling reaction D + HD → D2 + H in a pure solid HD matrix and in a D2 matrix with a 0.23% HD admixture at temperatures between 130 mK and 1.5 K. We found that the exchange reaction rates, kexHD ∼ 3 × 10-27 cm3 s-1 in the pure HD matrix, and kexD2 = 9(4) × 10-28 cm3 s-1 in the D2 matrix, are nearly independent of temperature within this range. This confirms the quantum tunnelling nature of these reactions, and their ability to proceed at temperatures down to absolute zero. Based on these observations we concluded that exchange tunneling reaction H + H2 → H2 + H should also proceed in a H2 matrix at the lowest temperatures. On the other hand, the recombination of H atoms in solid H2 and D atoms in solid D2 is substantially suppressed at the lowest temperatures as a result of a decreased probability of resonant tunneling of atoms when they approach each other.

Bose-Einstein condensation of magnons in atomic hydrogen gas.

We report on experimental observation of Bose-Einstein condensation (BEC)-like behavior of quantized electron spin waves (magnons) in a dense gas of spin-polarized atomic hydrogen. The magnons are trapped and controlled with inhomogeneous magnetic fields and described by a Schrödinger-like wave equation, in analogy to the BEC experiments with neutral atoms. We have observed the appearance of a sharp feature in the ESR spectrum displaced from the normal spin wave spectrum. We believe that this observation corresponds to a sudden growth of the ground-state population of the magnons and emergence of their spontaneous coherence for hydrogen gas densities exceeding a critical value, dependent on the trapping potential. We interpret the results as a BEC of nonequilibrium magnons which were formed by applying the rf power.

Dynamic nuclear polarization of high-density atomic hydrogen in solid mixtures of molecular hydrogen isotopes.

We report on magnetic resonance studies of high-density atomic hydrogen and deuterium in solid hydrogen matrices at temperatures below 1 K. Average concentrations of H atoms ≈3×10(19) cm(-3) are obtained in chemical tunneling reactions of isotope exchange with D atoms. The products of these reactions are closely located pairs of H atoms near D2 molecules with strong exchange interactions. We discovered a dynamic nuclear polarization effect on H atoms created by pumping the center of the H electron spin resonance spectrum, similar to the Overhauser effect in metals. Our results indicate that H atoms may be arranged inside molecular matrices at separations equivalent to local concentrations of 2.6×10(21) cm(-3). This opens up a way to build a metallic state of atomic hydrogen at zero pressure.

Guiding and trapping of electron spin waves in atomic hydrogen gas.

We present a high magnetic field study of electron spin waves in atomic hydrogen gas compressed to high densities of ∼10(18) cm(-3) at temperatures ranging from 0.26 to 0.6 K. We observed a variety of spin wave modes caused by the identical spin rotation effect with strong dependence on the spatial profile of the polarizing magnetic field. We demonstrate confinement of these modes in regions of strong magnetic field and manipulate their spatial distribution by changing the position of the field maximum.

A large octupole magnetic trap for research with atomic hydrogen.

We describe the design and performance of a large magnetic trap for storage and cooling of atomic hydrogen (H). The trap operates in the vacuum space of a dilution refrigerator at a temperature of 1.5 K. Aiming at a large volume of the trap, we implemented the octupole configuration of linear currents (Ioffe bars) for the radial confinement, combined with two axial pinch coils and a 3 T solenoid for the cryogenic H dissociator. The octupole magnet consists of eight race-track segments, which are compressed toward each other with magnetic forces. This provides a mechanically stable and robust construction with a possibility of replacement or repair of each segment. A maximum trap depth of 0.54 K (0.8 T) was reached, corresponding to an effective volume of 0.5 l for hydrogen gas at 50 mK. This is an order of magnitude larger than ever used for trapping atoms.

Rapid screening of commercially available herbal products for the inhibition of major human hepatic cytochrome P450 enzymes using the N-in-one cocktail.

Self-administration of complementary products concurrently with conventional medication is increasingly common. The potential for cytochrome P450 (CYP) inhibition requires investigation. The N-in-one assay with ten probe substrates for nine CYPs was used with human liver microsomes to investigate ten products. CYP inhibition was measured in a single liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis. Estimated IC(50)-values were determined for the extracts that produced significant inhibition (less than 100 microg ml(-1)). Inhibition of CYP2C19 by dong quai (IC(50) = 13.7-14.3 microg ml(-1) for the methanolic extract) and CYP2D6 by goldenseal (IC(50) = 6.7 and 6.3 microg ml(-1) for the aqueous and methanolic extracts, respectively), are of particular concern as the potential for adverse interactions is high. The inhibition of CYP2C8 by horsetail (IC(50) = 93 microg ml(-1) for the aqueous extract) requires further investigation, as the potential for concurrent use with products that require CYP2C8 for metabolism is significant. CYP3A4 inhibition varied depending on the probe reaction being monitored. The earlier reported findings of inhibition by black cohosh, goldenseal and gotu kola were confirmed. The present work has shown that the N-in-one cocktail is a rapid and reliable method that can be used as an initial screen to help prioritize products that require more detailed investigations and it can also be applied to monitor product variability.

Experimental cell with a Fabry-Pérot resonator tuned in situ for magnetic resonance studies of matrix-isolated radicals at temperatures below 1 K.

We describe the design and construction of an experimental cell for the study of free radicals in macroscopically thick films of solidified molecular and rare gases by 128 GHz Electron Spin Resonance (ESR) at temperatures below 1 K. The ESR resonator has an open Fabry-Pérot design, and its frequency can be tuned in situ by adjusting the spacing between the mirrors. The tuning mechanism consists of a piezo positioner and a stainless-steel edge-welded bellows, which can change the resonator frequency by at least 6 GHz. The films of solidified gases can be deposited either directly from a room temperature reservoir or by recondensing from a specially arranged chamber. The free radicals can be created in the solid films by dissociating matrix species by running an rf discharge in a helium vapor. We suggest that such a sample cell design can also be used for a broad range of low-temperature ESR experiments where sample cooling needs to be enhanced by the presence of superfluid helium.

Characterization of benzo(a)pyrene hydroxylase of trout liver.

Trout liver microsomes contained as 0.40 nmole of cytochrome P-450 per mg of protein and a NADPH-cytochrome c reductase activity of 23 nmoles of cytochrome c reduced per mg of protein per min at 22 degrees. Associated with these was a high benzo(a)pyrene hydroxylase activity, which required NADPH and O2 and was inhibited by CO. With thin-layer chromatography, at least five metabolites could be identified (including dihydrodiols, phenols, and quinones of benzo(a)pyrene). Inhibitors such as 2-diethylaminoethyl-2,2-diphenylvalerate, aminopyrine, metyrapone, pyridine, n-octylamine, and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane were relatively ineffective in inhibiting trout benzo(a)pyrene hydroxylase. Typical inhibitors of 3-methylcholanthrene-induced cytochrome (P-448), such as alpha-naphthoflavone, zoxazolamine, and testosterone, were effective, however. With benzo(a)pyrene it was possible to induce type I spectral change in trout cytochrome P-450. In spite of the many enzymatic characteristics of cytochrome P-448, trout cytochrome P-450 had maximum absorbance at 450.6 nm. when in reduced form and complexed with CO. the ethyl isocyanide gave an interaction spectrum with reduced trout liver cytochrome P-450 resembling that of control rat.

Role of cytochrome P450 2D6 (CYP2D6) in the stereospecific metabolism of E- and Z-doxepin.

The tricyclic antidepressant, doxepin, is formulated as an irrational mixture of E (trans) and Z (cis) stereoisomers (85%: 15%). We examined the stereoselective metabolism of doxepin in vitro, with the use of human liver microsomes, recombinant CYP2D6 and gas chromatography-mass spectrometry. In human liver microsomes over the concentration range 5-1500 microM, the rate of Z-doxepin N-demethylation exceeded that of E-doxepin above 100 microM in two of three livers. Eadie-Hofstee plots were curvilinear indicating the involvement of several enzymes in N-demethylation. Coincubation of doxepin with 7,8-naphthoflavone and ketoconazole reduced the rates of N-demethylation of E- and Z-doxepin by 30-50% and 40-60%, respectively, suggesting the involvement of CYP1A and CYP3A4, whilst quinidine had little effect on N-demethylation. In contrast, doxepin hydroxylation was exclusively stereo-specific; E-doxepin and E-N-desmethyldoxepin were hydroxylated with high affinity in liver microsomes and by recombinant CYP2D6 (Km in the range of 5-8 microM), but there was no evidence of Z-doxepin hydroxylation. In 'metabolic consumption' experiments with liver microsomes (having measurable CYP2D6 activity) and initial substrate concentration of 1 microM, the consumption of E-doxepin was greater (P < 0.05, n = 5) than that of Z-doxepin. Quinidine inhibited the consumption of E-doxepin but did not affect the consumption of Z-doxepin. With N-desmethyldoxepin, quinidine inhibited the consumption of E-N-desmethyl-doxepin whereas Z-N-desmethyldoxepin appeared to be a terminal oxidative metabolite. In summary, CYP2D6 is a major oxidative enzyme in doxepin metabolism; predominantly catalysing hydroxylation with an exclusive preference for the E-isomers. The relatively more rapid metabolism of E-isomeric forms, and the limited metabolic pathways for the Z-isomers may explain the apparent enrichment of Z-N-desmethyldoxepin that is observed in vivo.

Metabolism of dexfenfluramine in human liver microsomes and by recombinant enzymes: role of CYP2D6 and 1A2.

Dexfenfluramine has been widely used as an appetite suppressant in the treatment of obesity. It was recently shown that the apparent non-renal clearance of dexfenfluramine was significantly lower in poor metabolizers than in extensive metabolisers of debrisoquine which suggested the involvement of the polymorphically expressed enzyme, CYP2D6, in dexfenfluramine metabolism. In this study, human liver microsomes and yeast-expressed recombinant enzymes were used to examine dexfenfluramine metabolism in vitro. In human liver microsomes, the major product of dexfenfluramine was nordexfenfluramine with lesser amounts of a novel metabolite, N-hydroxynordexfenfluramine, and ketone and alcohol derivatives being formed. Eadie-Hofstee plots (v against v/[s]) of nordexfenfluramine formation between 1 and 1000 microM substrate concentration were biphasic in three of four liver microsome samples examined, with mean Km values of 3 and 569 microM for the high and low affinity enzymes, respectively. At a substrate concentration (0.5 microM) around the known therapeutic plasma concentration, there was negligible inhibition of microsomal dexfenfluramine N-dealkylation by sulphaphenazole and ketoconazole, but between 33 and 100% inhibition by quinidine, and 0-58% inhibition by 7,8-naphthoflavone in seven liver samples. In human liver microsomes, there was also a significant correlation (rs= 0.79, n = 10, P < 0.01) between dextromethorphan O-demethylation and dexfenfluramine (at 1 microM) N-dealkylation activities. Dexfenfluramine was a specific inhibitor (IC50 46 microM) of CYP2D6-mediated dextromethorphan O-demethylation in human liver microsomes but did not appreciably inhibit six other cytochrome P450 isoform-selective activities for CYP1A2, 2A6, 2C9, 2C19, 2E1 and 3A activities in human liver microsomes. Yeast-expressed recombinant human CYP2D6 metabolized dexfenfluramine with high affinity (Km 1.6 microM, Vmax 0.18 nmol min(-1) nmol P450(-1)) to nordexfenfluramine which was the sole product observed. Recombinant CYP1A2 was a lower affinity enzyme (Km 301 microM, Vmax 1.12 nmol min(-1) nmol P450(-1)) and produced nordexfenfluramine with small amounts of N-hydroxynordexfenfluramine. This is the first detailed study to examine the in-vitro metabolism of dexfenfluramine in human liver microsomes and by recombinant human P450s. We were able to identify CYP2D6 (high affinity) and CYP1A2 (low affinity) as the major enzymes catalysing the N-dealkylation of dexfenfluramine in human liver microsomes.

Indacrinone: modification of diuretic, uricosuric, and kaliuretic actions by amiloride.

The response to indacrinone, a new indanone diuretic, was studied in 12 healthy subjects. Ten milligrams alone and in combination with either 2.5 mg or 5 mg amiloride was given in a randomized double-blind study with placebo control to study its action and to assess the optimum combination. Indacrinone alone induced an increase in urine flow rate and in sodium, potassium, and hydrogen ion excretion for at least 8 hr. Indacrinone also induced an initial uricosuria in the first 4 hr, followed by urate retention in the subsequent 12 to 24 hr, with no resultant change in the mean 24-hr urate excretion and minimal changes in the serum urate concentrations. The addition of 2.5 mg amiloride to the 10 mg indacrinone lowered potassium excretion to control levels, whereas addition of 5 mg amiloride resulted in net retention of potassium. With both doses of amiloride, the increased free hydrogen ion excretion after indacrinone returned to placebo levels. There were minor increases in serum creatinine, consistent with volume depletion due to the diuresis. There was a reduction in urinary calcium excretion. Our study shows that the combination of 10 mg indacrinone and 2.5 mg amiloride induces useful diuresis with minimal overall effect on urate, potassium, and hydrogen ion excretion.

Characterization of a marsupial glutathione transferase, a class Alpha enzyme from Brown Antechinus (Antechinus stuartii).

The major form of glutathione transferase from the marsupial Antechinus stuartii has been purified and characterized as an Alpha class enzyme (Ast GST A1-1) with distant sequence relationships to other class Alpha sublines, compatible with the early origin of marsupials. Amino acid replacements toward the closest enzyme characterized (chicken, form A3) involve no less than 79 positions (36%). At the active site, as deduced from comparisons with the known tertiary structure of the corresponding human enzyme, over half of the residues (8 of 15) ascribed to substrate binding interactions are exchanged although the general character of that site is conserved, while only 1 of 11 positions ascribed to interactions with GSH is exchanged. Class variability and species variability appear to coincide, with divergent segments centering around positions 33-49, 103-130 and 205-222. The pattern is reminiscent of that in similarly multiple MDR alcohol dehydrogenases. Both these enzyme families involved in cellular defense reactions have diverged considerably.

Inhibition of soluble glutathione S-transferase by diuretic drugs.

Glutathione transferases are believed to play an important protective role in the various tissues of animals and man by catalysing the glutathione conjugation of electrophilic drugs and electrophilic drug metabolites. Many of these compounds have the potential to react with vital cellular macromolecules in the absence of this enzyme system. We have investigated the interaction of a number of high ceiling diuretics with the glutathione transferases contained in the cytosolic fraction of the rat liver. Of bumetanide, ethacrynic acid, furosemide, indacrynic acid and tienilic acid, only ethacrynic acid was conjugated with glutathione. Further experiments revealed that ethacrynic, indacrynic and tienilic acids are all potent inhibitors of glutathione S- aryltransferase . Glutathione S- alkyltransferase and glutathione S-epoxide transferase were also inhibited by the diuretics, but to a lesser extent than glutathione S- aryltransferase . The diuretics giving the greatest inhibition of these reactions were chemically related to ethacrynic acid. The concept where inhibition of glutathione-S-transferase by a drug may enhance its own toxicity is considered. This mechanism has also the potential of enhancing the toxicity of other concurrently administered drugs which normally require glutathione S-transferase for detoxication.

Inhibition of purified rat liver glutathione S-transferase isozymes by diuretic drugs.

Seven soluble rat liver glutathione S-transferase isozymes were isolated and the inhibition of these isozymes by selected diuretics was investigated using 1-chloro-2,4-dinitrobenzene as substrate. All isozymes were inhibited to some extent under the experimental conditions used, but there was significant isozyme dependent selectivity of inhibition. The greatest inhibitory effect (over 80%) was found when the phenoxyacetic acid diuretics and indacrynic acid were incubated with glutathione S-transferase 3-3, 3-4 and 4-4. The sulphamoylbenzoic acid diuretics, furosemide and bumetanide, were found to have a lesser effect on the isozymes studies. As glutathione S-transferase are thought to play an important protective role in the various tissues of animals and man, by catalysing the glutathione conjugation of electrophilic drugs and drug metabolites, their inhibition may be toxicologically important.

Glutathione transferases and glutathione-binding proteins of termites: purification and characterisation.

Termites have an important role in the cycling of carbon and trace elements in the biosphere through their degradation of wood, grasses and humus. Glutathione transferases (GSTs) are detoxication enzymes found in all organisms; GSTs are known to protect insects from the toxic effects of plant chemicals and pesticides. The activities and characteristics of termite GSTs were investigated in three Australian termite families. Multiple GST isozymes were purified from whole body preparations of the three termite species by affinity chromatography and subsequent chromatofocusing. Termite GSTs exhibited a broad range of activities toward model substrates but were most active with 1-chloro-2,4-dinitrobenzene. The pI values of termite GSTs ranged between 7.4 and 5.8, while the apparent molecular weights of subunits ranged between 25.9 and 27.7 kDa. The N-terminal sequence of a glutathione-binding protein that was purified from Mastotermes darwiniensis was similar to the N-terminal sequence of a streptococcal protein of unknown function previously implicated in glomerulonephritis in humans.

The effect of orally administered viable probiotic and dairy lactobacilli on mouse lymphocyte proliferation.

Four common Lactobacillus strains were screened for their effects on proliferation of mouse splenic lymphocytes. Mice received perorally 10(9) viable bacteria kg(-1) body weight for 7 days. Lactobacillus acidophilus treatment enhanced ex vivo basal proliferation (by 43%) and B-cell response at suboptimal and optimal concentrations of lipopolysaccharide (LPS) (by 27-28%). Conversely, Lactobacillus casei, Lactobacillus gasseri and Lactobacillus rhamnosus inhibited both basal proliferation (by 14-51%) and mitogen-stimulated lymphoproliferation, particularly at supra-optimal concentrations of concanavalin A (by 43-68%) and LPS (by 23-62%). Therefore, these Lactobacillus strains demonstrate strain-specific effects on B- and T-cells and may also alter the splenocyte sensitivity to the cytotoxic effects of mitogens.