Alternaria alternata NADP-dependent mannitol dehydrogenase is an important fungal allergen.

BACKGROUND: Alternaria alternata is one of the most important allergenic fungi worldwide. Mannitol dehydrogenase (MtDH) has previously been shown to be a major allergen of Cladosporium herbarum and cross-reactivity has been demonstrated for several fungal allergens. OBJECTIVE: The present study's objective was to clone the MtDH from an A. alternata cDNA library, express and purify the recombinant non-fusion protein and test its IgE-binding properties. Methods A cDNA library prepared from A. alternata hyphae and spores was screened for mannitol dehydrogenase by DNA hybridization with the radioactively labelled C. herbarum homologue as a probe. The resulting clone was sequenced and heterologously expressed in Escherichia coli as a recombinant non-fusion protein, which was purified to homogeneity and analysed for its IgE-binding capacity. RESULTS: The coding sequence of the full-length cDNA clone comprises 798 bp encoding a protein with a molecular mass of 28.6 kDa and a predicted pI of 5.88. Protein sequence analysis revealed an identity of 75% and a homology of 86% between the MtDHs of A. alternata and C. herbarum. The functional mannitol dehydrogenase was expressed in the E. coli strain BL21(DE3) transformed with the vector pMW172 and purified to homogeneity. The enzyme catalyses the NADPH-dependent conversion of d-fructose to d-mannitol. In IgE-ELISA and immunoblots, MtDH is recognized by 41% of A. alternata-allergic patients. In vivo immunoreactivity of the recombinant MtDH was verified by skin prick testing. Finally, inhibition-ELISA experiments confirmed cross-reactivity between the MtDHs of A. alternata and C. herbarum. CONCLUSION: Mannitol dehydrogenase (Alt a 8) represents an important new allergen of the ascomycete A. alternata that might be suitable for improving diagnostic and therapeutic procedures.     

Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E

BACKGROUND: The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen-specific IgE. OBJECTIVE: To test the performance of this allergen microarray in a serological analytical study. METHODS: Standard allergens contained in grass pollen (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) and tree pollen (Bet v 1 and Bet v 2) were used as a model system. The detection of allergen-specific serum IgE using microarrays was compared with standard test systems: CAP/RAST and an in-house ELISA. In order to test the analytical sensitivity of the assays, geometric dilutions of a serum pool containing high levels of pollen-specific IgE from allergic individuals were tested in each system. To assess the analytical specificity, the sera of 51 patients with presumptive allergic symptoms were collected before diagnosis. Thereafter, the results for grass/tree-pollen-specific IgE were compared. RESULTS: The microarray has a good dynamic range similar to the CAP/RAST system. Microarray and ELISA showed comparable analytical sensitivity exceeding the CAP/RAST system. With respect to the analytical specificity, no significant cross-reactivity of the allergens was observed. For two of the allergens tested, weak positive signals were detected in the microarray test system, whereas they were not detectable by CAP/RAST. CONCLUSION: A good correlation of presently used methods to detect serum IgE and the novel microarray test system was observed. As a next step, a careful validation of this method for a multitude of allergens and a thorough clinical evaluation has to be provided. Microarray testing of allergen-specific IgE can be presumed to be the method of choice for a prospective component-resolved diagnosis of Type I allergy, and the basis for the design and monitoring of a patient-tailored specific immunotherapy in the future.     

Factors influencing the onset of ouabain inhibition of Na,K-ATPase from guinea-pig myocardium.

1. The onset of ouabain inhibition was quantified by analysis with an integrated rate equation from experiments in which the activity of Na,K-ATPase from guinea-pig myocardium had been altered with adenosine 5'-triphosphate (ATP, 0.3-9 mmoll-1) in the absence and presence of a detergent. 2. Under control conditions with increasing ouabain (0.1-100 mumoll-1) and ATP (0.3-1 mmoll-1) concentrations, inhibition developed faster. The acceleration by ouabain became less effective at saturating concentrations leading to a non-linear relationship between pseudo-first-order rate constants of inhibition and ouabain concentration. With a rise of ATP to 3 and 9 mmoll-1, i.e., near total Mg concentration (5 mmoll-1), inhibition was retarded presumably because the free concentrations of Mg and uncomplexed ATP changed. Varying the ATP concentration had little effect on ouabain potency at steady state; Hill coefficients were less than 1. 3. The detergent alamethicin (23 micrograms ml-1) neither interfered with Na,K-ATPase activity nor with inhibition at steady state but accelerated its onset. This supports a role for a lipid barrier in the development of inhibition. 4. While the reaction of low concentrations of ouabain with the receptors seemed to govern inhibition rate, with an increase in steroid concentration in the presence of alamethicin, ATP-dependent enzyme activity interfered with the onset of inhibition. The transition of the enzyme between ouabain-sensitive and ATP-hydrolytic conformations consequently causes the non-linear concentration-dependence of pseudo-first-order rate constants. As the Hill coefficient was less than 1, a reaction of ouabain with two receptors also could have contributed to the special concentration-dependence of inhibition rates.     

Magnetic resonance imaging (MRI) of the breast

Zusammenfassung Grundlagen: Der Zweck war es, die Anzahl der im Mammogramm entdeckten duktalen Carcinoma in situ (DCIS) und multizentrische Karzinome aufzudecken. Die verschiedenen Methoden der Biopsie werden diskutiert. Methodik: Rastermammographie (Fokus 0,3 mm) und hochaufl?sende Sonographie (7,5 bis 10 MHz) wurden bei Brustkrebspatientinnen eingesetzt. Ergebnisse: Der Zeitraum der aufgelisteten Patientinnen erstreckt sich über 20 Jahre. Die Zahl der DCIS hat in den letzten Jahren zugenommen und betr?gt nun 18%. Die Mammographie ist hoch sensitiv für Entdeckung von Mikrokalzifikationen. Die Spezifit?t ist gering. Schlu?folgerungen: Die Anzahl der entdeckten DCIS ist in den letzten Jahren besonders angestiegen und wird auch noch weiter bei Verbesserung der mammographischen Techniken ansteigen. Diese Studie wurde vom Ludwig-Boltzmann-Institut für radiologisch-physikalische Tumordiagnostik unterstützt.     

Stereospecific hydroxylation of (+)-sparteine (pachycarpine) in the rat

Pachycarpine (4), the optical antipode of the lupine alkaloid (-)-sparteine (1), has been prepared from (-)-lupanine; its metabolism was studied in rats. After isolation and chromatographic purification, streochemically homogeneous (+)-(4S)-hydroxysparteine (7) was identified as the major urinary metabolite by use of mass spectrometry and high-field NMR-spectroscopy.     

Motor evoked responses recorded epidurally in a patient with Guillain-Barré syndrome

Summary The case of a 75-year-old man with Guillain-Barré syndrome is presented. By means of transcranial electrical stimulation and epidural recording at the spinal level L2-3, distinct potentials with a latency of 21ms were obtained when the patient was tetraplegic. At the same time electromyographic responses of the thenar and anterior tibial muscles were absent following both transcranial and peripheral nerve stimulation. The patient recovered partially within 4 weeks. It is concluded that epidurally recorded motor evoked responses allow electrophysiological assessment of the descending pathways even in severe cases of Guillain-Barré syndrome and might contribute to a more accurate prediction of outcome.     

The evaluation of run-off prior to infra-inguinal bypass reconstruction - a modified scoring system based on flow measurement.

OBJECTIVES: To compare angiographic scoring and flow measurements in the assessment of run-off prior infra-inguinal bypass. PATIENTS AND METHODS: In a series of 108 consecutive infra-inguinal bypasses, run-off was scored on the basis of pre- and post-operative angiograms and related to intra- and post-operative flow rates as determined by Doppler ultrasonography. RESULTS: There was a highly significant correlation between the angiographic score and flow (p = 0.0000), as well as between angiographic score (p = 0.0000), flow (p = 0.0000) and the level of distal anastomosis. Flow determined per crural vessel (quotient of flow to angiographic score) proved to be independent of the level of distal bypass anastomosis (p = 0.20). CONCLUSION: In this study, angiographic scoring and Doppler flow measurements were equally valid means for the assessment of run-off. Our system allows an objective assessment of run-off independently of the distal bypass anastomosis level and provides a functional estimation of run-off.