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.

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.

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.

Integrative plasmid vector for constructing single-copy reporter systems to study gene regulation in Rhizobium meliloti and related species.

The integrative system of phage 16-3 of Rhizobium meliloti 41 was shown to function in several bacterial species belonging to the Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium genera. It might also function in many other bacterial species provided that both the target site (attB) and the required host factor(s) are present. Here we report on the construction of a new integrative vector that can be utilized in gene regulation studies. It provides an opportunity to create a single-copy set-up for characterizing DNA-protein interactions in vivo, in a wide range of bacteria. To demonstrate the usefulness of the vector, transcription repression by binding of the C repressor protein of phage 16-3 to wild type operators was studied. The assay system provided highly reproducible quantitative data on repression.

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.

Footprinting studies of specific complexes formed by RepA, a replication initiator of plasmid pCU1, and its binding site.

The basic replicon of plasmid pCU1 contains three different replication origins. Replication initiated from the oriB origin requires pCU1-encoded protein RepA. Previously, information analysis of 19 natural RepA binding sequences predicted a 20-bp sequence as a RepA binding site. Guanines contacting RepA in the major groove of DNA have also been determined. In this study, we used the missing-nucleoside method to determine all of the bases relevant to RepA binding. The importance of some thymine bases was also confirmed by a missing-thymine site interference assay. Participation of the 5-methyl groups of two thymines (at positions -6 and 7) in RepA binding was pointed out by a missing-thymine methyl site interference assay. Phosphate groups of the DNA backbone which strongly interfered with RepA binding upon ethylation were also identified. The pattern of contacting positions mapped by hydroxyl radical protection footprinting indicates that RepA binds to one face of B-form DNA. The length of the binding site was found to be 20 bp by dissociation rate measurement of complexes formed between RepA and a variety of binding sequences. The symmetry of the binding site and that of the contacting bases, particularly the reacting 5-methyl groups of two thymines, suggest that pCU1-encoded RepA may contact its site as a homodimer.

Identification of site-specific recombination genes int and xis of the Rhizobium temperate phage 16-3.

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.

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.

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.

Family themes: transmission and transformation.

This article describes a method for doing therapy that uses multisystemic themes that combine meaning and action to facilitate therapeutic change. By identifying central themes that operate at the individual, dyadic, triadic, whole family, intergenerational, and sociocultural levels, the therapist is able to develop effective interview questions and design useful interventions. In this method, behavioral symptoms are framed as a current manifestation of an overarching theme. This orientation enables family and therapist to de-pathologize symptoms and work collaboratively toward change. Case examples from a wide variety of families with differing presenting problems, interactional patterns, three-generational histories, and cultural backgrounds, illustrate the efficacy of the method.

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.

[A comparison of 2 methods of plastic cast fixation in treatment of loco classico radius fracture. A prospective, randomized study]. / Ein Vergleich von 2 Gipsfixationsmethoden bei der Behandlung der Radiusfraktur loco classico. Eine prospektive, randomisierte Studie.

The purpose of this study was to compare the functional and radiological result of two different positions of the wrist in a plaster cast following Colles' fracture. For this prospective study, each of 50 patients with type A 2.2, A 3.3, C 1.2 or C 2.2 (AO classification) fractures of the radius was randomly assigned to one of two groups. Both groups were treated in the same way as far as anaesthesia and reduction were concerned. The only difference in treatment lay in the position of fixation in plaster. In group 1 the wrist was immobilized in neutral flexion-extension. In group 2 the wrist was dorsiflexed 20 degrees, while the carpus was pushed in a volar direction by an impression in the plaster cast. At review 2-7 years after the accidents, the two groups were compared with reference to symptoms, range of motion at the wrist, power of first closure and radiographic appearance. In group 1 there were 5 patients with significant disability, compared with only 1 in group 2. A significant difference was found in the range of movement between the two groups for flexion and ulnar abduction (p < 0.01). The loss of power of first clenching (difference between injured and healthy hand) was 6.2 mmHg for group 1 and 3.8 mmHg for group 2 (not significant). The radiographic examination showed significant differences both in sagittal inclination (p < 0.001) and in radial shortening (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Negative control of plasmid DNA replication by iterons. Correlation with initiator binding affinity.

Initiation of DNA replication and negative control of initiation frequency in many bacterial plasmids are mediated by multiple binding sites (iterons) for the replicon-encoded initiator protein. Here we show that a single iteron of plasmid P1, when cloned in a multicopy vector, pUC19, can control replication of a miniP1 plasmid effectively. Using this assay system, several iterons with single point mutations were analyzed. The degree of control was directly correlated with the binding affinity of the iterons for the initiator protein in vitro. The control was also unaffected whether or not a wild type iteron was flanked with strong transcription terminators. We conclude that initiator binding is the only activity of P1 iteron sequences relevant to replication control.

Missing-base and ethylation interference footprinting of P1 plasmid replication initiator.

RepA, the replication initiator protein of plasmid P1, binds to specific 19 bp sequences on the plasmid DNA. Earlier footprinting studies with dimethylsulfate identified the guanines that contact RepA through the major groove of DNA. In this study, base elimination was used to identify the contribution of all four bases to the binding reaction. Depurination and depyrimidation of any base in the neighborhood of the contacting guanines was found to decrease RepA binding. These results are consistent with the notion that RepA contacts bases of two consecutive major grooves on the same face of DNA. We also observed that depurination but not methylation of three guanines (G3, G8 and G9) affected binding. We identified the DNA phosphate groups (3 in the top strand, one of which mapped between G8 and G9, and 4 in the bottom strand, one of which was adjacent to C3) that strongly interfered with RepA binding upon ethylation. These results indicate that certain bases (e.g. G3, G8 and G9) may not contact RepA directly but contribute to base and backbone contacts by maintaining proper structure of the binding site.

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.

The bacterial attachment site of the temperate Rhizobium phage 16-3 overlaps the 3' end of a putative proline tRNA gene.

Bacteriophage 16-3 inserts its genome into the chromosome of Rhizobium meliloti strain 41 (Rm41) by site-specific recombination. The DNA regions around the bacterial attachment site (attB) and one of the hybrid attachment sites bordering the integrated prophage (attL) were cloned and their nucleotide sequences determined. We demonstrated that the 51 bp region, where the phage and bacterial DNA sequences are identical, is active as a target site for phage integration. Furthermore it overlaps the 3' end of a putative proline tRNA gene. This gene shows 79% similarity to the corresponding proline tRNA-like genomic target sequence of certain integrative plasmids in Actinomycetes.

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.