Inhomogeneous high harmonic generation in krypton clusters.

High order harmonic generation from clusters is a controversial topic: conflicting theories exist, with different explanations for similar experimental observations. From an experimental point of view, separating the contributions from monomers and clusters is challenging. By performing a spectrally and spatially resolved study in a controlled mixture of clusters and monomers, we are able to isolate a region of the spectrum where the emission purely originates from clusters. Surprisingly, the emission from clusters is depolarized, which is the signature of statistical inhomogeneous emission from a low-density source. The harmonic response to laser ellipticity shows that this generation is produced by a new recollisional mechanism, which opens the way to future theoretical studies.

High-harmonic transient grating spectroscopy of NO2 electronic relaxation.

We study theoretically and experimentally the electronic relaxation of NO(2) molecules excited by absorption of one ∼400 nm pump photon. Semiclassical simulations based on trajectory surface hopping calculations are performed. They predict fast oscillations of the electronic character around the intersection of the ground and first excited diabatic states. An experiment based on high-order harmonic transient grating spectroscopy reveals dynamics occurring on the same time scale. A systematic study of the detected transient is conducted to investigate the possible influence of the pump intensity, pump wavelength, and rotational temperature of the molecules. The quantitative agreement between measured and predicted dynamics shows that, in NO(2), high harmonic transient grating spectroscopy encodes vibrational dynamics underlying the electronic relaxation.

Efficient generation of near infra-red single photons from the zero-phonon line of a single molecule.

Using the zero-phonon line (ZPL) emission of a single molecule, we realized a triggered source of near-infra-red (lambda = 785 nm) single photons at a high repetition rate. A Weierstrass solid immersion lens is used to image single molecules with an optical resolution of 300 nm (approximately 0.4lambda) and a high collection efficiency. Because dephasing of the transition dipole due to phonons vanishes at liquid helium temperatures, our source is attractive for the efficient generation of single indistinguishable photons.

Binding of nitroxide stearate spin labels to bovine serum albumin.

1. 12-Nitroxide stearate binds to bovine serum albumin at about four independent and equivalent binding sites with an association constant of about 10(6) M-1. The binding at these high affinity binding sites is significantly reduced by addition of unlabeled stearate. These data suggest that nitroxide stearates probe the high affinity binding sites for long-chain fatty acids. 2. Qualitative analyses of the ESR spectra of 5-, 12- and 16-nitroxide stearate bound to bovine serum albumin and measurements of the interaction of these compounds so bound with ferricyanide ion provide a rough description of the binding site as follows: the polar headgroup of the spin-labeled fatty acid is rigidly fixed, but fairly accessible to paramagnetic ions. The middle part of the hydrocarbon chain of bound stearate spin label also is rigidly fixed but differs in being shielded from the solvent, presumably by a hydrophobic cleft. The methyl terminus shows greater motion, appearing to move within a narrow cone, and also appears to be somewhat accessible to paramagnetic ions.

EPR titration of ovine prostaglandin H synthase with hemin.

To characterize further the prosthetic group of PGH synthase (EC 1.14.99.1), titrations of the apoenzyme with hemin were investigated by EPR. The first hemin bound per polypeptide showed an EPR signal at g = 6.7 and 5.3 (rhombicity 9%) and was tentatively assigned to the hemin effective as prosthetic group of PGH synthase. Additional hemin bound showed a less rhombic signal (g = 6.3 and 5.8, rhombicity 3%) presumably due to nonspecific hydrophobic binding sites not effective in catalysis.

Metabolic denitrosation of diphenylnitrosamine: a possible bioactivation pathway.

Nitrosodiphenylamine was tested for induction of DNA single strand breaks in rat hepatocytes and Chinese hamster V 79 cells with the alkaline filter elution assay. While in rat hepatocytes DNA damage was observed, negative results were obtained in V 79 cells. In view of the metabolic capacity of hepatocytes and the chemical structure of nitrosodiphenylamine it seems likely that cytochrome P-450-dependent, reductive denitrosation might be necessary for exerting this effect. Therefore the metabolism of nitrosodiphenylamine was investigated in phenobarbital-induced mouse liver microsomes and some of the metabolites were also tested. One metabolite was identified as diphenylamine whereas the others were identified as a ring-hydroxylated derivative of diphenylamine and its corresponding quinoneimine. Diphenylhydroxylamine which was not detected in the microsomes as a metabolite produced a significant amount of DNA single strand breaks in V 79 cells. When diphenylhydroxylamine was incubated with microsomes electron spin resonance spectrum was observed which indicated the formation of the diphenylnitroxide radical. This radical seems to be mediated by auto-oxidation rather than by enzymatic catalysis. Whether diphenylhydroxylamine might be responsible for the observed genetoxic effects of nitrosodiphenylamine assumed to be produced via active oxygen species is discussed.

Development of a biosensor for E. coli based on a flexural plate wave (FPW) transducer.

To fulfill the need for rapid, cost-effective and sensitive methods for the detection of bacteria in medical diagnostics, food technology, biotechnology and environmental monitoring, a development of a bacterial sensor was initiated. Our approach of a biosensor for E. coli is based on an acousto-gravimetric flexural plate wave (FPW) transducer (gravimetric detection limit of less than 6 ng in a 32 microns thick sensitive layer in aqueous media), and an immunoaffinity layer on the transducer membrane for the molecular recognition of the target bacteria. An intermediate layer of covalently coupled poly (acrylic acid) yielded a major reduction of the non-specific binding to the metal surface. Such a biosensor, using antibodies against E. coli K12 and E. coli 15 outer surface antigens, yielded a detection range of 3.0 x 10(5) to 6.2 x 10(7) cells/ml for samples with the corresponding bacteria. To increase the sensitivity further, an amplification method using microspheres coupled with antibodies against E. coli was tested as a sandwich assay, and up to now a five-fold amplification of the signal has been achieved.

A biohybrid system to interface peripheral nerves after traumatic lesions: design of a high channel sieve electrode.

Peripheral nerve lesions lead to nerve degeneration and flaccid paralysis. The first objective in functional rehabilitation of these diseases should be the preservation of the neuro-muscular junction by biological means and following functional electrical stimulation (FES) may restore some function of the paralyzed limb. The combination of biological cells and technical microdevices to biohybrid systems might become a new approach in neural prosthetics research to preserve skeletal muscle function. In this paper, a microdevice for a biohybrid system to interface peripheral nerves after traumatic lesions is presented. The development of the microprobe design and the fabrication technology is described and first experimental results are given and afterwards discussed. The technical microprobe is designed in a way that meets the most important technical requirements: adaptation to the distal nerve stump, suitability to combine the microstructure with a containment for cells, and integrated microelectrodes as information transducers for cell stimulation and monitoring. Micromachining technologies were applied to fabricate a polyimide-based sieve-like microprobe with 19 substrate-integrated ring electrodes and a distributed counter electrode. Monolithic integration of fixation flaps and a three-dimensional shaping technology led to a device that might be adapted to nerve stumps with neurosurgical sutures in the epineurium. First experimental results of the durability of the shaping technology and electrochemical electrode properties were investigated. The three-dimensional shape remained quite stable after sterilization in an autoclave and chronic implantation. Electrode impedance was below 200 kOmega at 1 kHz which ought to permit recording of signals from nerves sprouting through the sieve holes.

The kinetics of binding of o-methyl red to the outer surface of unilamellar spherical phospholipid vesicles.

The kinetics of adsorption of the proton carrier o-methyl red to the surface of unilamellar spherical phospholipid vesicles have been investigated by means of the temperature-jump relaxation technique with absorbance detection. Single-exponential relaxation curves were observed with time constants in the range 30-130 microseconds. o-Methyl red binds in both its anionic form A- and protonated form AH. Adsorption-desorption of the two species is coupled by two fast protolytic reactions, occurring in the aqueous bulk phase and in the surface region of the membrane. The rate constants for adsorption and desorption of the two species were obtained from the dependences of the relaxation time on lipid concentration at different pH values. The analysis yielded apparent adsorption rate constants of kasAH = 9.8 X 10(6) M-1 s-1 and kasA = 1.3 X 10(6) M-1 s-1 (expressed in terms of monomeric lipid), and kasAH = 1.2 X 10(11) M-1 s-1 and kasA = 1.6 X 10(10) M-1 s-1 (expressed in terms of vesicle concentration). From the order of these rate constants it is concluded that adsorption of both species is actually diffusion-controlled. The peculiar pH dependence of the relaxation time is a consequence of the protolytic reaction in the surface region of the membrane. Its implication for the kinetics of adsorption-desorption processes are discussed.

Influence of vesicle curvature on fluorescence relaxation kinetics of fluorophores.

The effect of membrane curvature on the fluorescence decay of 2-p-toluidinyl-naphthalene-6-sulfonic acid (TNS), 2-(9-anthroyloxy) stearic acid (2-AS) and 12-(9-anthroyloxy)-stearic acid (12-AS) was investigated for egg lecithin vesicles of average diameter dm = 22 nm and 250 nm. The biexponential fluorescence decay of TNS at the red edge of the emission spectrum was analysed according to the model of Gonzalo and Montoro [1]. Over the entire temperature range (1-40 degrees C) the small TNS labelled vesicles showed significantly shorter solvent relaxation times tau(r) than their larger counterparts (e.g. 1.3 ns compared with 2.1 ns at 5 degrees C), indicating a higher mobility of the hydrated headgroups in the highly curved, small vesicles. The fluorescence decay of both AS derivatives is also biexponential. While the shorter decay times (1-3 ns) are practically identical for small and large vesicles, the longer decay times (5-14 ns) are identical only for 12-AS but not for 2-AS. This indicates that the microenvironment is similar in small and large vesicles deep in the membrane in spite of the differences in curvature.

Models for ferric cytochrome P450. Characterization of hemin mercaptide complexes by electronic and ESR spectra.

Hemin coordinated with mercaptide sulfur as fifth ligand and various sixth ligands were investigated as models for cytochrome P450 in its native ferric low-spin state and its ligand complexes. Mixing the hemin with its ligands below -60 degrees C prevented the reduction of the hemin by mercaptide and made it possible to characterize each sample both by electronic and ESR spectra. Excess of mercaptide formed hemin-dimercaptide complexes with hyperporphyrin spectra with two Soret bands around 380 and 370 nm. The second mercaptide could be exchanged by other ligands with hydroxyl, phosphine, thioether, isocyanide, amine, imidazole, and pyridine groups. The comparison of these spectral data with cytochrome P450 substantiates mercaptide as the fifth ligand and makes a hydroxyl group a more likely candidate for the native sixth ligand than an imidazole group.

Active site-specific reconstituted copper(II) horse liver alcohol dehydrogenase: a biological model for type 1 Cu2+ and its changes upon ligand binding and conformational transitions.

Insertion of Cu2+ ions into horse liver alcohol dehydrogenase depleted of its catalytic Zn2+ ions creates an artificial blue copper center similar to that of plastocyanin and similar copper proteins. The esr spectrum of a frozen solution and the optical spectra at 296 and 77 K are reported, together with the corresponding data for binary and ternary complexes with NAD+ and pyrazole. The binary complex of the cupric enzyme with pyrazole establishes a novel type of copper proteins having the optical characteristics of Type 1 and the esr parameters of Type 2 Cu2+. Ternary complex formation with NAD+ converts the Cu2+ ion to a Type 1 center. By an intramolecular redox reaction the cuprous enzyme is formed from the cupric enzyme. Whereas the activity of the cupric alcohol dehydrogenase is difficult to assess (0.5%-1% that of the native enzyme), the cuprous enzyme is distinctly active (8% of the native enzyme). The implications of these findings are discussed in view of the coordination of the metal in native copper proteins.

Binding of thiols to microsomal cytochrome P-450.

Lipophilic thiol compounds interact spectrally with liver microsomes from phenobarbital-pretreated rats by formation of unusual optical difference spectra with peaks at 378, 471, 522 and 593 nm in the oxidized state. The binding kinetics were biphasic. The EPR spectrum of cytochrome P-450 was slightly modified but the magnitude of the low-spin signal was unchanged. n-Octanethiol competitively displaced metyrapone and n-octane from the active site of cytochrome P-450. Other thiols behaved similarly with variations in the magnitude and the affinity of the binding process. Tertiary thiols caused the formation of the high-spin cytochrome P-450 substrate complex, and model studies with myoglobin revealed that steric hindrance prevented the liganding of the tertiary thiol group to the ferric cytochrome P-450. Addition of thiols to dithionite reduced microsomes resulted in relatively small spectral changes with maxima at 449 nm typical for ligand complexes of the ferrous cytochrome. It was concluded that lipophilic thiols can be bound as ligands by at least two species of oxidized cytochrome P-450 which represent, however, not more than about one fifth of the total cytochrome P-450 content in liver microsomes from phenobarbital-pretreated rats.

Binding of nitrosamines to cytochrome P-450 of liver microsomes.

The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.

Fast and precise positioning of single cells on planar electrode substrates.

For cell biosensors and for studying neural networks using planar electrode substrates, a suitable technique for positioning single cells on electrodes was needed. We reported a new method for fast and efficient positioning of single cells on ring electrodes by controlled suction through holes. We described the microfabrication of electrode substrates with microholes and the cell positioning procedure. L929 cells and Neuro 2A cells could be positioned in parallel without cell damage.

A comparison of butoconazole nitrate cream with econazole nitrate cream for the treatment of vulvovaginal candidiasis.

In a randomized, single-blind, parallel study the safety and efficacy of 2% butoconazole nitrate cream used for 3 days were compared with those of 1% econazole nitrate cream used for 7 days at night in patients with vulvovaginal candidiasis. Patients in both treatment groups had positive potassium hydroxide smears and fungal cultures, and were similar in age, disease duration and history, obstetric history and contraceptive use. Of the 75 patients enrolled, 63 with a Candida albicans infection were included in the efficacy analyses. Evaluations were made at the start of the study (visit 1), after 10-23 days (visit 2) and after 24-45 days (visit 3). Both drugs significantly reduced all signs and symptoms, and at visits 2 and 3 the percentages of patients considered microbiologically, clinically and therapeutically cured were consistently higher for butoconazole- than for econazole-treated patients, although differences were not statistically different. Although both drugs were safe and well tolerated, it is concluded that butoconazole because of its much shorter regimen and superior clinical and microbiological performance was the drug of choice.

Asymmetric reconstitution of the erythrocyte anion transport system in vesicles of different curvature: implications for the shape of the band 3 protein.

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent nonaethylene glycol lauryl ether and then reconstituted in spherical egg phosphatidylcholine bilayers as described earlier (U. Scheuring, K. Kollewe, W. Haase, and D. Schubert, J. Membrane Biol. 90, 123-135 (1986)). The resulting paucilamellar proteoliposomes of average diameter 70 nm were transformed into smaller vesicles by French press treatment and fractionated according to size by gel filtration. The smallest protein-containing liposomes obtained had diameters around 32 nm; still smaller vesicles were free of protein. All proteoliposome samples studied showed a rapid sulfate efflux which was sensitive to specific inhibitors of band 3-mediated anion exchange. In addition, the orientation of the transport protein in the vesicle membranes was found to be "right-side-out" in all samples. This suggests that the orientation of the protein in the vesicle membranes is dictated by the shape of the protein's intramembrane domain and that this domain has the form of a truncated cone or pyramid.

Cimetidine and ranitidine: their interaction with human and pig liver microsomes and with purified cytochrome P-450.

Cimetidine and ranitidine interact with microsomes from human and pig liver and with purified cytochrome P-450 in the ligand-type manner. The affinity for cimetidine is about 10 times as high as that for ranitidine. Accordingly amplitudes of the specta are much higher for cimetidine. These results are in accordance with those obtained earlier with rat liver microsomes. The inhibitory potency of either compound with regard to dealkylation of 7-ethoxycoumarin appears to be less in the human preparation.

[Post-partum contraception (author's transl)]. / Contraception du post partum.

The authors have come to the conclusion that the ideal time to start female contraception is when the periods return following delivery. They arrived at this conclusion after studying the disadvantages of the various methods of hormone contraception and of mechanical female contraception immediately following delivery. Directly after delivery contraception should be in the hands of the man and not the woman. If better information is given to the couple during pregnancy and in the postnatal period it should direct them towards such formations of contraception. The problem, however, of grand multipara who are feckless, ignorant and of low social class, still remains. Medroxyprogesterone, if it is used at all immediately after delivery, should be given in a dose of 150 mg. As far as tubal ligation by mini-laparotomy after delivery goes this is permanent contraception and its indications should be considered with very great care.

[Plasma levels of HCS and beta SP1 in normal and pathological pregnancies]. / Dosage plasmatique de HCS et betaSP1 dans les grossesses normalies et pathologiques.

A study was carried out on 599 pregnant women from whom 2,991 levels were estimated of HCS and beta SP1 to determine their variations. The control group was of 400 normal pregnancies (1,770 couples of levels of HCS and beta SP1. The pathological population was in two groups: - non-complicated pathological pregnancies, from whom 510 coupled levels were estimated in 77 women, and - complicated pregnancies, from whom 711 coupled levels were estimated in 122 patients. This pathological group was compared with the control population statistically. The results conform to those that have already been published. This work shows two original features: - very high levels of beta SP1 and HCS ;are an index of the severity of the prognosis of fetuses and hydramnios, - a drop in the level of beta SP1 towards the end of pregnancies is a warning that fetal distress may occur in labour.