Dissociative electron attachment and electronic excitation in Fe(CO)5.

In a combined experimental and theoretical study we characterize dissociative electron attachment (DEA) to, and electronically excited states of, Fe(CO)5. Both are relevant for electron-induced degradation of Fe(CO)5. The strongest DEA channel is cleavage of one metal-ligand bond that leads to production of Fe(CO)4-. High-resolution spectra of Fe(CO)4- reveal fine structures at the onset of vibrational excitation channels. Effective range R-matrix theory successfully reproduces these structures as well as the dramatic rise of the cross section at very low energies and reveals that virtual state scattering dominates low-energy DEA in Fe(CO)5 and that intramolecular vibrational redistribution (IVR) plays an essential role. The virtual state hypothesis receives further experimental support from the rapid rise of the elastic cross section at very low energies and intense threshold peaks in vibrational excitation cross sections. The IVR hypothesis is confirmed by our measurements of kinetic energy distributions of the fragment ions, which are narrow (∼0.06 eV) and peak at low energies (∼0.025 eV), indicating substantial vibrational excitation in the Fe(CO)4- fragment. Rapid IVR is also revealed by the yield of thermal electrons, observed in two-dimensional (2D) electron energy loss spectroscopy. We further measured mass-resolved DEA spectra at higher energies, up to 12 eV, and compared the bands observed there to resonances revealed by the spectra of vibrational excitation cross sections. Dipole-allowed and dipole/spin forbidden electronic transitions in Fe(CO)5-relevant for neutral dissociation by electron impact-are probed using electron energy loss spectroscopy and time-dependent density functional theory calculations. Very good agreement between theory and experiment is obtained, permitting assignment of the observed bands.

Dissociative electron attachment to 2,4,6-trichloroanisole and 2,4,6-tribromoanisole molecules.

2,4,6-trichloroanisole and 2,4,6-tribromoanisole were investigated by means of electron transmission spectroscopy and two different types of dissociative electron attachment spectrometers. The results obtained were interpreted with the support of density functional theory calculations. The dominant dissociative decay channels of the temporary molecular negative ions lead to the formation of Cl- and Br- in the low electron energy region. Formation of long-lived parent anions is observed at thermal electron energies. Their relative intensity depends on the experimental time window, ∼36 µs in the case of the static magnet mass analyzer and ∼200 µs for the quadrupole mass analyzer employed. The results obtained may be useful for rapid detection of these compounds in wine and pharmaceutical industries, as well as other branches connected to the food industry, e.g., packaging.

Electron ionization and dissociation of aliphatic amino acids.

We present experimental and theoretical study of electron ionization and dissociative ionization to the gas phase amino acids valine, leucine, and isoleucine. A crossed electron/molecular beams technique equipped with quadrupole mass analyzer has been applied to measure mass spectra and ion efficiency curves for formation of particular ions. From experimental data the ionization energies of the molecules and the appearance energies of the fragment ions were determined. Ab initio calculations (Density Functional Theory and G3MP2 methods) were performed in order to calculate the fragmentation paths and interpret the experimental data. The experimental ionization energies of parent molecules [P](+) 8.91 ± 0.05, 8.85 ± 0.05, and 8.79 ± 0.05 eV and G3MP2 ionization energies (adiabatic) of 8.89, 8.88, and 8.81 eV were determined for valine, leucine, and isoleucine, respectively, as well as the experimental and theoretical threshold energies for dissociative ionization channels. The comparison of experimental data with calculations resulted in identification of the ions as well as the neutral fragments formed in the dissociative reactions. Around 15 mass/charge ratio fragments were identified from the mass spectra by comparison of experimental appearance energies with calculated reaction enthalpies for particular dissociative reactions.

Thermal desorption effects on fragment ion production from multi-photon ionized uridine and selected analogues.

Experiments on neutral gas-phase nucleosides are often complicated by thermal lability. Previous mass spectrometry studies of nucleosides have identified enhanced relative production of nucleobase ions (e.g. uracil+ from uridine) as a function of desorption temperature to be the critical indicator of thermal decomposition. On this basis, the present multi-photon ionization (MPI) experiments demonstrate that laser-based thermal desorption is effective for producing uridine, 5-methyluridine, and 2'-deoxyuridine targets without thermal decomposition. Our experiments also revealed one notable thermal dependence: the relative production of the sugar ion C5H9O4 + from intact uridine increased substantially with the desorption laser power and this only occurred at MPI wavelengths below 250 nm (full range studied 222-265 nm). We argue that this effect can only be rationalized plausibly in terms of changing populations of different isomers, tautomers, or conformers in the target as a function of the thermal desorption conditions. Furthermore, the wavelength threshold behavior of this thermally-sensitive MPI channel indicates a critical dependence on neutral excited state dynamics between the absorption of the first and second photons. The experimental results are complemented by density functional theory (DFT) optimizations of the lowest-energy structure of uridine and two further conformers distinguished by different orientations of the hydroxymethyl group on the sugar part of the molecule. The energies of the transitions states between these three conformers are low compared with the energy required for decomposition.

Electron impact ionization of furanose alcohols.

Electron impact ionization of the gas phase 3-furanol, tetrahydro (3-hydroxytetrahydrofuran, 3HTHF) and 2-furanmethanol, tetrahydro (alpha-tetrahydrofurfuryl alcohol, THFA) molecules has been studied both experimentally and theoretically. The electron induced positive ion formation has been investigated experimentally using a crossed electron/neutral beams technique in combination with a quadrupole mass spectrometry. The mass spectra of both molecules have been determined at the incident electron energy of 70 eV. The ionization efficiency curves for each parent cation and a number of fragment cations have been measured near the threshold, and the corresponding appearance energies have been derived using an iterative fitting procedure based on the Wannier threshold law, taking into account the incident electron energy resolution. The appearance energies of the parent cations were experimentally determined to be (9.620+/-0.058) eV for (C(4)H(8)O(2)(+)/3HTHF) and (9.43+/-0.12) eV for (C(5)H(10)O(2)(+)/THFA), which are in a good agreement with G3MP2 calculated results: 9.480 and 9.419 eV, respectively. The most abundant cations in the mass spectra were determined to be 57 amu for 3HTHF and 71 amu for THFA, with the corresponding experimentally determined appearance energies of (10.22+/-0.10) eV and (9.574+/-0.062) eV, respectively. With the help of the energies calculated at B3LYP and G3MP2 levels of theory, the possible fragmentation patterns were discussed.

Determination of the binding sites of RepA, a replication initiator protein of the basic replicon of the IncN group plasmid pCU1.

A 2kb DNA region of the broad-host-range plasmid pCU1 carries all of the information essential for the stable maintenance of the plasmid and to express the same host-range specificity. It was predicted that the protein required to initiate replication from at least one of the three origins of the plasmid is encoded by the longest open-reading frame (ORF239) of the three overlapping in-frame ORFs located within the 2 kb region. The product of ORF239 has been named RepA. The initiator protein was overexpressed, purified and used for in vitro binding studies. Gel mobility shift experiments were performed to localize RepA binding sites. The DNA sequence protected by the bound RepA molecule(s) was determined by DNase I footprinting and 19 of a 20 bp long sequence that is part of the protected sequence were located in two clusters flanking the repA gene. A plasmid created by linking a 310 bp fragment (nucleotides 238 to 547) of the 2 kb region to the antibiotic resistance genes carried by the omega fragment, can be maintained stably if the RepA protein is supplied in trans. We conclude that this 310 bp DNA fragment, which consists of a short G+C and a long A+T rich region and the cluster of five RepA binding sites, carries a functional origin of the plasmid-protein dependent replication. The position of this origin indicates that it is oriB, one of the three origins previously identified by electron microscopy. The second cluster of RepA binding sites is downstream of the repA gene and consists of 14 sites that are in inverted orientation compared with the binding sites located in the oriB region. They are part of the region that was shown formerly to be involved in controlling the copy number of the plasmid. In contrast to oriB, binding of RepA to neither the oriS nor oriV region was detected.

Information analysis of sequences that bind the replication initiator RepA.

The replication initiator protein RepA of plasmid P1 can bind to 14 sites on the plasmid. These sites are variously used to autoregulate RepA synthesis and for initiation and control of DNA replication. Analysis of information (degree of conservation) at the sites revealed three sequence patches of high conservation. By saturation mutagenesis, the conservation at the outer two patches was found to contribute to RepA binding more critically. The guanine bases that are likely to contact RepA through the major groove were identified by methylation interference and methylation protection experiments. These bases mapped to the outer two patches and were separated by one turn of the helix. Therefore, they belong to major grooves on the same face of DNA. All backbone contacts of the protein, determined by hydroxyl radical footprinting, also mapped to the same face. We conclude from this that RepA binds to its site on one face of the DNA. Information analysis of binding sites for several prokaryotic repressors and activators, where the nature of DNA-protein contacts are known, revealed a correlation between the positions of high conservation and the positions of major grooves that faced the protein. The middle patch of high conservation in the RepA binding sites is an exception since in this region a minor groove is likely to face the protein. The simplest model for minor groove contacts suggests that in B-form DNA a T.A base-pair cannot easily be distinguished from an A.T pair by inspection of the minor groove. Yet in the RepA site, a T-->A mutation in the middle patch significantly affects binding. Therefore, the simplest models for both minor and major groove contacts are unlikely. It is possible that the patch determines the proper conformation of the site and thereby contributes to recognition indirectly.

Activation of DNA binding by the monomeric form of the P1 replication initiator RepA by heat shock proteins DnaJ and DnaK.

RepA protein of plasmid P1 binds to arrays of 19 bp repeat sequences (iterons) and mediates initiation of replication and its control. Escherichia coli heat shock proteins DnaJ and DnaK can stimulate iteron binding activity of RepA in an ATP-dependent fashion. It has been proposed that RepA binds to DNA as monomers and that the stimulation in binding involves monomerization of RepA dimers which are inactive in the binding reaction. RepA-iteron and RepA-RepA interactions have been measured in this study to determine the equilibrium constants of the two reactions. The apparent KD value for RepA-iteron binding decreased from 10 nM to no more than 0.2 nM at increasing concentrations of the heat shock proteins. The stimulation of binding appears to be due to an increase in active RepA fraction and not to a change in the maximum binding capacity of the active species. This view was deduced from measurements of active RepA fraction, which increased in the presence of heat shock proteins, and from measurements of dissociation rate constants, which were independent of the heat shock protein concentrations. Accounting for the active fractions, the true KD value was estimated to be 0.10(+/- 0.09) nM in 20 mM Tris.HCl (pH 8), 100 mM NaCl, 40 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, ATP (50 microM), bovine serum albumin (50 micrograms/ml), calf thymus DNA (50 micrograms/ml) and glycerol (5%). The dissociation rate constant was 1.5 x 10(-2) s-1 and the calculated association rate constant was 1.5 x 10(8) M-1 s-1. Ultracentrifugation analyses of RepA at 15,000 r.p.m. in the above buffer but without ATP, bovine serum albumin, calf thymus DNA and glycerol, revealed that the protein was in monomer-dimer equilibrium with a KD of 2.6(+/- 0.2) microM at 5 degrees C. Therefore, at protein concentrations used in the binding reactions, RepA is monomeric (> 99.5%), in confirmation of the earlier result that RepA binds as a monomer. It follows that the species that is stimulated to bind by the heat shock proteins is also a monomeric form of RepA.

Kidney transplantation in recipients with mental retardation: clinical results in a single-center experience.

Mental retardation has been a controversial relative contraindication to organ transplantation. Currently, there are few data available in the literature that describe the outcome of kidney transplantation in mentally retarded patients. In a series of 1,271 kidney transplantations performed between January 1968 and March 1996, we identified eight patients (0.6%) with significant mental retardation (IQ < 70). Only cooperative patients supervised by a reliable long-term caregiver, with long life expectancy, and able to take medication under supervision, were accepted as candidates, independent of the IQ level. At a mean follow-up of 7.3 years, seven patients are alive with functioning grafts, and one lost the kidney to chronic rejection 10 years after transplantation and died of sepsis after resuming dialysis. The 1- and 5-year patient and graft survival are thus 100%. Compliance with immunosuppressive treatment and clinical follow-up was excellent in all of the recipients. The patient quality of life and health were judged by the support persons as highly improved after transplantation in comparison to dialysis. We conclude that kidney transplantation in properly selected patients with mental retardation provides excellent patient and graft survival rates and improves quality of life. In such patients, the presence of mental retardation should not be considered a contraindication to kidney transplantation.

Integrative promoter cloning plasmid vectors for Rhizobium meliloti.

A set of integrative 'promoter probe' plasmids were constructed for both translational and transcriptional fusions. The vectors are based on the broad host range, low copy number plasmid pRK290 (IncPl) in which the attachment site of Rhizobium phage 16-3 and the lacZ gene of Escherichia coli were combined. The vectors integrate into the chromosome of Rhizobium meliloti, providing also the advantages of the single copy promoter probe cassettes. Thus they fulfil the prerequisite of the systems used for investigating gene regulation. The plasmids were applied for the study of the transcription regulation of the 16-3 phage. Their versatile use is also demonstrated.

Modulation of the phototoxic effect of hypericin in human leukemia CEM cell line by N-ethylmaleimide, amiloride and omeprazole.

Hypericin is a photosensitizing plant pigment from Hypericum perforatum with multiple modes of light-induced biological activities due to production of singlet oxygen and/or excited-state proton transfer with consequent pH drop in the hypericin environment. In the present work, we studied the effects of three inhibitors of crucial mechanisms responsible for intracellular pH (pHi) regulation on hypericin phototoxicity: N-ethylmaleimide (NEM), an inhibitor of H+-ATPase, 5'-(N,N-dimethyl)-amiloride (DMA), an inhibitor of Na+/H+ exchanger, and omeprazole (OME), an inhibitor of H+K+-ATPase. Our experiments show that the effect of hypericin at 1 x 10(-5) and 1 x 10(-6) mol x l(-1) was significantly potentiated by NEM (1 x 10(-7)-1 x 10(--9) mol x l(-1)) and DMA (1 x 10(-6) and 1 x 10(-7) mol x l(-1)) in leukemic CEM cell line. On the other hand, OME had no significant effect on hypericin cytotoxicity. Our results support the hypothesis that the excited-state proton transfer and the consequent acidification of hypericin environment could play a role in the biological activity of hypericin.

Staging reciprocal metaphors in a couples group.

This paper has described a format for treating couples using the technique of couples choreography to define the marital relationship in metaphorical terms. When the metaphors are acted out, the reciprocity in the relationship is translated into concrete images. These images provide the basis for systemic interventions aimed at disrupting the escalating reciprocity. Prechange tests are used to regulate the speed of change in relation to the reciprocal positions. Change is viewed as an unsettling phenomenon that temporarily unbalances the marital relationship. The group serves as a theatrical setting in which the marital relationships are "staged" and examined with humor and objectivity. An atmosphere of experimentation is created, which is necessary for carrying out the unconventional tasks.