Measurement of polarization observables T, P, and H in π0 and η photoproduction off quasi-free nucleons.

The target asymmetry T, recoil asymmetry P, and beam-target double polarization observable H were determined in exclusive π0 and η photoproduction off quasi-free protons and, for the first time, off quasi-free neutrons. The experiment was performed at the electron stretcher accelerator ELSA in Bonn, Germany, with the Crystal Barrel/TAPS detector setup, using a linearly polarized photon beam and a transversely polarized deuterated butanol target. Effects from the Fermi motion of the nucleons within deuterium were removed by a full kinematic reconstruction of the final state invariant mass. A comparison of the data obtained on the proton and on the neutron provides new insight into the isospin structure of the electromagnetic excitation of the nucleon. Earlier measurements of polarization observables in the γp→π0p and γp→ηp reactions are confirmed. The data obtained on the neutron are of particular relevance for clarifying the origin of the narrow structure in the ηn system at W=1.68GeV. A comparison with recent partial wave analyses favors the interpretation of this structure as arising from interference of the S11(1535) and S11(1650) resonances within the S11-partial wave.

Observation of the pη

Data on the beam asymmetry Σ in the photoproduction of η mesons off protons are reported for tagged photon energies from 1130 to 1790 MeV (mass range from W=1748 MeV to W=2045 MeV). The data cover the full solid angle that allows for a precise moment analysis. For the first time, a strong cusp effect in a polarization observable has been observed that is an effect of a branch-point singularity at the pη^{'} threshold [E_{γ}=1447 MeV (W=1896 MeV)]. The latest BnGa partial wave analysis includes the new beam asymmetry data and yields a strong indication for the N(1895)1/2^{-} nucleon resonance, demonstrating the importance of including all singularities for a correct determination of partial waves and resonance parameters.

[Acute generalized exanthematous pustulosis: first case associated with a Chlamydia pneumoniae infection]. / Pustulose exanthématique aiguë généralisée: premier cas décrit en relation avec une infection à Chlamydia pneumoniae.

UNLABELLED: Skin reactions to drugs or infections can be very important. CASE REPORT: A 14-year-old boy developed fever, a diffuse pustular rash and a respiratory distress. Chest X-rays and serology were consistent with an acute Chlamydia pneumoniae infection. A skin biopsy revealed an acute generalized exanthematous pustulosis. CONCLUSION: Acute generalized exanthematous pustulosis (AGEP) is a disease manifested by a diffuse eruption of follicular sterile pustules. Most of the cases are drug induced, but they may be secondary to viral or bacterial infections. We report the first case of AGEP in a child with an acute C. pneumoniae infection.

A database search for hammerhead ribozyme motifs.

The hammerhead ribozyme is the smallest naturally occurring RNA endonuclease. It is found in subviral plant pathogens and transcripts of satellite DNA from a limited number of organisms. We have performed a database search for novel examples of this catalytic RNA, taking into consideration the recently defined structural requirements for an efficient cleavage under physiological magnesium ion concentrations. In this search, we find, apart from the known examples, several hundreds of motifs in organisms of all kingdoms of life. In a first set of experiments, we analysed hammerhead ribozymes from Arabidopsis thaliana. We found that these sequences are tissue-specifically expressed and that they undergo self-cleavage in planta. Furthermore, their activity under physiological magnesium ion concentrations depends on functional loop-loop interactions, as shown by the lack of activity of appropriate mutants.

Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR.

We have used (19)F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5fluorouridine. We have studied the chemical shift and linewidths of (19)F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg(2+). The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg(2+) with an association constant in the range of 100 to 500 M(-1), depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range.

Thermodynamics of ion-induced RNA folding in the hammerhead ribozyme: an isothermal titration calorimetric study.

The hammerhead ribozyme undergoes a well-defined two-stage conformational folding process, induced by the binding of magnesium ions. In this study, we have used isothermal titration calorimetry to analyze the thermodynamics of magnesium binding and magnesium ion-induced folding of the ribozyme. Binding to the natural sequence ribozyme is strongly exothermic and can be analyzed in terms of sequential interaction at two sites with association constants K(A) = 480 and 2840 M(-1). Sequence variants of the hammerhead RNA give very different isothermal titration curves. An A14G variant that cannot undergo ion-induced folding exhibits endothermic binding. By contrast, a deoxyribose G5 variant that can undergo only the first of the two folding transitions gives a complex titration curve. However, despite these differences the ITC data for all three species can be analyzed in terms of the sequential binding of magnesium ions at two sites. While the binding affinities are all in the region of 10(3) M(-1), corresponding to free energies of Delta G degrees = -3.5 to -4 kcal mol(-1), the enthalpic and entropic contributions show much greater variation. The ITC experiments are in good agreement with earlier conformational studies of the folding of the ion-induced folding of the hammerhead ribozyme.

Evaluation of uranyl photocleavage as a probe to monitor ion binding and flexibility in RNAs.

In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in five RNA molecules with known X-ray structures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA(Phe) and tRNA(Asp) from yeast. Uranyl photocleavage was observed at specific positions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl(2). Uranyl photocleavage correlates well with sites of low calculated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in flexible and open to the solvent regions that can undergo induced-fit without disrupting the native fold. Uranyl photocleavage was compared to N-ethyl-N-nitrosourea and lead-induced cleavages in the context of the three-dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at flexible and accessible sites and correlate with the unspecific cleavages prevalent in dynamic and open regions. Applied in a magnesium-dependent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-affinity metal ion environments, as well as regions involved in conformational transitions.

[Spondylodiscitis due to Propionibacterium acnes. Case report and review of the literature]. / Spondylodiscite à Propionibacterium acnes. A propos d'un cas et revue de la littérature.

Propionibacterium acnes, a gram positive anaerobic organism, is a component of normal skin flora. It can exceptionally be a source of osteoarticular infection (osteitis, arthritis, spondylodiscitis). We report a case of Propionibacterium acnes spondylodiscitis following lumbar puncture. This observation should alert the clinician to the fact that Propionibacterium acnes may rarely cause spondylodiscitis and lumbar pain, and should be considered a causative agent of bone infections after local procedures.

Generation and application of asymmetric hammerhead ribozymes.

Most researchers who intend to suppress a particular gene are interested primarily in the application of ribozyme technology rather than its mechanistic details. This article provides some background information and describes a straightforward strategy to generate and test a special design of a ribozyme: the asymmetric hammerhead ribozyme. This version of a hammerhead ribozyme carries at its 5' end the catalytic domain and at its 3' end a relatively long antisense flank that is complementary to the target RNA. Asymmetric hammerhead ribozymes can be constructed via polymerase chain reaction amplification, and rules are provided on how to select the DNA oligonucleotides required for this reaction. In addition to details on construction, we describe how to test asymmetric hammerhead ribozymes for association with the target RNA in vitro, so that RNA constructs can be selected and optimized for fast hybridization with their target RNA. This test can allow one to minimize association problems caused by the secondary structure of the target RNA. Additionally, we describe the in vitro cleavage assay and the determination of the cleavage rate constant. Testing for efficient cleavage is also a prerequisite for reliable and successful application of the technology. A carefully selected RNA will be more promising when eventually used for target suppression in living cells.

Expression of CD28 and CD38 by CD8+ T lymphocytes in HIV-1 infection correlates with markers of disease severity and changes towards normalization under treatment. The Swiss HIV Cohort Study.

The relationship between blood CD8+ T lymphocyte subsets, as defined by CD28 and CD38 expression, and plasma viraemia and CD4+ T cells in HIV-1 infection was investigated. In a cross-sectional study of 46 patients with either no or stable anti-retroviral treatment, there was a strong negative correlation between the percentage of CD8+CD28- and the percentage of CD4+ T cells (r = -0.75, P < 0.0001), and a positive correlation between absolute numbers of CD8+CD28+ and CD4+ T cells (r = 0.56, P < 0.0001). In contrast, the expression of CD38 by CD8+ T lymphocytes correlated primarily with plasma viraemia (e.g. the percentage of CD38+ in CD8bright cells, r = 0.76, P < 0.0001). In the 6 months following triple therapy initiation in 32 subjects, there was a close correlation between changes (delta) in CD8+CD28+ or CD8+CD28- and in CD4+ T cells (e.g. delta % CD8+CD28+ versus delta % CD4+, r = 0.37, P = 0.0002; delta % CD8+CD28- versus delta % CD4+, r = -0.66, P < 0.0001). A marked decline of the number of CD8+ T cells expressing CD38 was also observed. These results suggest the existence of a T cell homeostasis mechanism operating in blood with CD4+ and CD8+CD28+ cells on the one hand, and with CD8+CD28- cells on the other. In addition, the percentage of CD38+ cells in CD8+ cells, generally considered an independent prognostic factor, could merely reflect plasma viral load.

Length variation of helix III in a hammerhead ribozyme and its influence on cleavage activity.

The previously described HIV-1 directed hammerhead ribozyme 2as-Rz12 can form with its target RNA 2s helices I and III of 128 and 278 base pairs (bp). A series of derivatives was made in which helix III was truncated to 8, 5, 4, 3, and 2 nucleotides (nt). These asymmetric hammerhead ribozymes were tested for in vitro cleavage and for inhibition of HIV-1 replication in human cells. Truncation of helix III to 8 bp did not affect the in vitro cleavage potential of the parental catalytic antisense RNA 2as-Rz12. Further truncation of helix III led to decreased cleavage rates, with no measurable cleavage activity for the 2 bp construct. All catalytically active constructs showed complex cleavage kinetics. Three kinetic subpopulations of ribozyme-substrate complexes could be discriminated that were cleaved with fast or slow rates or not at all. Gel purification of preformed ribozyme-substrate complexes led to a significant increase in cleavage rates. However, the complex cleavage pattern remained. In mammalian cells, the helix III-truncated constructs showed the same but no increased inhibitory effect of the comparable antisense RNA on HIV-1 replication.

Myocarditis of mixed connective tissue disease: favourable outcome after intravenous pulsed cyclophosphamide.

A 30-year-old woman with mixed connective tissue disease was admitted with Wernicke's aphasia and progressive dyspnoea with chest pain. Multiple brain infarcts on a computed tomographic scan were compatible with a thromboembolic aetiology. Echocardiography showed marked hypokinesia of the posterior wall, biventricular dilatation and a decreased left-ventricle ejection fraction (40%). A diagnosis of myocarditis was made on myocardial biopsies disclosing interstitial lymphocytic infiltrates and myocardial fibre necrosis. A treatment with steroids and monthly pulsed cyclophosphamide was introduced. The heart function rapidly improved as assessed by a left-ventricle ejection fraction of 55% and remained stable 17 months thereafter.

Crystal structures of modified apo-His117Gly and apo-His46Gly mutants of Pseudomonas aeruginosa azurin.

The X-ray crystal structures of two metal ligand mutants of azurin from Pseudomonas aeruginosa have been solved. In both mutants (His117Gly and His46Gly azurin) one of the copper coordinating histidine residues is replaced by a glycine, creating an empty space in the coordination sphere of the copper ion. The crystal structure of His117Gly azurin at 2.4 A resolution showed that this mutant had undergone partial oxidation at the disulfide bridge between Cys3 and Cys26 and full oxidation at the copper ligand Cys112. There is no copper present in the crystallized form and the bulky group of the oxidized cysteine at position 112 causes large structural rearrangements in the protein structure, especially in the loops connecting the beta-sheets. In the structure of the wild-type holo-azurin from P. aeruginosa the hydrophobic patch is important for the packing of the azurin molecules into dimers which then arrange into tetramers. The completely different packing of the apo-His117Gly mutant can be explained by the disruption of the hydrophobic patch area by the mutation-induced main-chain conformational change of residues 112 to 115. The structure of apo-His46Gly azurin at 2.5 A resolution is the same as the wild-type structure except for the immediate environment at the site of the mutation. In the His46Gly structure water molecules are found at positions that in the wild-type structure are occupied by the imidazole ring of His46 and the copper ion. The imidazole ring of His117 is shifted by about 1 A towards the surface of the protein, similar to that observed for 50% of the molecules in the wild-type apo-azurin structure. This shift causes a slight rearrangement of the monomers within the tetramer such that one local dyad becomes a crystallographic dyad parallel to the c-axis. This leads to a change in the space group from P2(1)2(1)2(1) to P2(1)2(1)2.

A spermidine-induced conformational change of long-armed hammerhead ribozymes: ionic requirements for fast cleavage kinetics.

The catalytic activity of the trans cleaving hammerhead ribozyme 2as-Rz12, with long antisense flanks of 128 and 278 nt, was tested under a wide range of different reaction conditions for in vitro cleavage of a 422 nt RNA transcript derived from human immunodeficiency virus type 1 (HIV-1). Depending on the reaction conditions, in vitro cleavage rates varied by a factor of approximately 100. Increasing concentrations of magnesium up to 1 M were found to enhance the reaction. Sodium when added simultaneously with magnesium showed an inhibitory effect on the cleavage reaction. Addition of sodium during pre-annealing, however, produced a stimulating effect. It was found that the additional inclusion of spermidine during pre-annealing further increased the reaction rate markedly. In accordance with accelerated cleavage, it was possible to identify a distinct, spermidine-induced conformer of the ribozyme-substrate complex. Under the most favourable conditions cleavage rates of 1/min were obtained, which are in the range of rates obtained for conventional hammerhead ribozymes with short antisense flanks. A comparison of thermodynamic data for short- and long-armed hammerhead ribozymes suggested that the activation entropy became unfavourable when helices I and III formed a long chain ribozyme-substrate complex. We conclude that in the absence of spermidine folding into the active conformation is impaired by increased friction of long helices, resulting in relatively low cleavage rates in vitro.

Comparison of effects of topical levocabastine and nedocromil sodium on the early response in a conjunctival provocation test with allergen.

BACKGROUND: Multiple ocular challenges or seasonal trials have demonstrated the efficacy of levocabastine and nedocromil sodium in the treatment of allergic conjunctivitis. OBJECTIVE: This study was designed to compare the protective effect of levocabastine eye drops with that of nedocromil in a conjunctival provocation test with allergen. METHODS: Twenty-four patients with allergic conjunctivitis to grass pollen were recruited. After a preliminary provocation to determine conjunctival reaction threshold (erythema of at least 50% of the conjunctiva with ocular itching), patients were randomized to receive either topical levocabastine (0.05%) or nedocromil (2%) 15 minutes before provocation. Erythema and pruritus intensity were recorded at each concentration of allergen up to the reaction threshold. RESULTS: The allergen concentration level necessary to reach reaction threshold was increased (p < 0.001) after treatment with both drugs. Comparison between screening and each treatment indicated that the shift in allergen concentration was significantly greater after levocabastine treatment than after nedocromil treatment (p = 0.019). Conjunctival itching (symptom score) and erythema (percent conjunctival surface) were also better controlled by levocabastine than by nedocromil during provocation (p < 0.05). CONCLUSION: In a provocation test with allergen, levocabastine and nedocromil were both effective in increasing the conjunctival tolerance to allergen, with better protection provided by levocabastine.

X-ray crystal structure of the two site-specific mutants Ile7Ser and Phe110Ser of azurin from Pseudomonas aeruginosa.

The blue copper protein azurin from Pseudomonas aeruginosa contains a single Trp residue that is believed to be involved in the inducible intramolecular electron transfer from a disulphide group to the copper centre. This residue shows in fluorescence spectra the highest energy emission of tryptophan-containing compounds at room temperature, which is explained by its rigid and highly hydrophobic environment. In order to investigate the role of the Trp residue in electron transfer and the influence of its environment, two mutations (17S and F110S) were introduced that were thought to increase the polarity and the mobility in its environment. The crystal structures of these mutants were solved at 2.2 A and 2.3 A resolution, respectively. These provide a structural basis for the changes observed in fluorescence spectra compared with the wild-type protein. We conclude from our results that these changes are not caused by a change in the dynamics of the Trp residue itself, but exclusively by an increased effective dielectric constant of the microenvironment of Trp48 and by changes in mobility of the mutated residues.

[Horton's disease: ophthalmological and neurological complication]. / Maladie de horton: complications ophtalmologiques et neurologiques.

We report the case of a 74-year-old women admitted to the hospital because of rapidly developing diplopia. The admission anamnesis revealed fronto-temporal headaches and pain in the maxilla for three days. A cerebral cT scan was normal, and a corticosteroid therapy was initiated. Diagnosis of Horton's syndrome was confirmed by the good response to treatment and by biopsy of the temporal artery. Different ocular and neurologic complications of Horton's disease, their clinical appearances and therapeutic measures are discussed by the authors.

[Anaphylactic shock]. / Le choc anaphylactique.

Anaphylactic shock is an unexpected, sudden and sometimes deadly event that attacks the patient in 75% of the cases without pre-existent history of allergy. In general, drugs, hymenopteric poisons and nutrients (according to the recent concept) are responsible. Beside the classical IgE-mediated anaphylactic shock there exists another anaphylactic shock, identical in its clinical picture and treatment but not in the mechanism of development. Epinephrine is the only effective drug in case of respiratory (bronchial asthma, laryngeal edema) or cardiovascular (hypotension, arrhythmias, hypovolemic shock) manifestation. It has to be administered as rapidly as possible.

Structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa at 2.2 A resolution.

The structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa has been determined by difference Fourier synthesis using phases from the wild-type azurin model. The final crystallographic R value is 0.170 for 17 394 reflections to a resolution of 2.2 A. The mutant crystallized in the orthorhombic space group P2(1)2(1)2(1), a = 57.4, b = 80.4, c = 110.3 A. The four molecules in the asymmetric unit are packed as a dimer of dimers. The nickel metal site of this mutant structure is similar to the zinc metal site in the azurin Asp47 mutant. The site-specific mutation was performed at residue Trp48, which is located in the center of the protein in a highly hydrophobic environment, to investigate its suggested role in the long-range electron-transfer pathway between the disulfide bond on one side of the protein to the Cu centre. The structure around the mutation site Met48 showed no significant change compared with the wild-type structure.