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.     

Formaldehyde in cryoprotectant propanediol and effect on mouse zygotes

Cryopreservation of human zygotes and embryos has been routinely performed by in-vitro fertilization clinics for many years. Karran and Legge (1996) first reported that formaldehyde (FA) present in the cryoprotective solutions can have a deleterious effect on mouse oocytes. FA is a cytotoxic, carcinogenic and mutagenic chemical. The effect of FA on mouse zygotes was investigated. In addition, the concentrations of FA in propanediol (PROH) obtained from various sources were determined. Pooled 1-cell embryos were dispensed into droplets of modified Ham's F10 or human tubal fluid containing various concentrations of FA. Since bovine serum albumin (BSA) may minimize toxicity additional trials were done as above in the absence of BSA. FA concentration in the standard 1.5 M PROH, from different sources in water, was measured in the same assay using a standard curve of 0-100 microM FA. FA in a complex medium had a significant deleterious effect on embryo development and hatching but only at 1 mM concentration (P < 0.000001; see Tables I-III). There was no significant effect of FA at 100 microM. However, in a simple medium even 50 microM FA decreased embryo hatching. FA was present in 1.5 M PROH from different sources (range 1.0-35.3 microM concentration). It appears that FA concentrations do not increase with storage because FA concentrations were low even after opening and storage for 3 years on the shelf. This suggests that FA is a contaminant during the manufacturing process and may vary from manufacturer to manufacturer and batch to batch. Until further studies are done to confirm the lack of toxicity to embryos during cryopreservation (with or without FA scavengers) it may be prudent to screen all batches of cryoprotectants for FA as part of quality control.      

Nachweis und Lokalisation von Porphyrinen im Nierenbiopsiegewebe bei chronischer hepatischer Porphyrie (Porphyria cutanea tarda) mittels Fluorescenzmikroskopie

Zusammenfassung Bei 3 Patienten mit chronischer hepatischer Porphyrie wurden erstmals Nierenbiopsiecylinder histologisch mittels Fluorescenzmikroskopie auf das Vorkommen von Porphyrinen untersucht. Porphyrinablagerungen wurden in den Epithelien der Hauptstücke und der angrenzenden Henleschen Schleifen gefunden, während Sammelrohre und Glomerula frei von Porphyrinen waren. Es wird diskutiert, ob aktive Sekretion oder Resorptionsphänomene die Porphyrinablagerungen in bestimmten Tubulusabschnitten bedingen, oder ob lediglich eine passive Imbibition des Gewebes (mit Porphyrinen) vorliegt.

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Summary In three patients with chronic hepatic porphyria (porphyria cutanea tarda) kidney biopsy specimens were investigated histologically by fluorescence microscopy. Typical red fluorescence indicating porphyrin deposits was found in the epithelial cells of convoluted renal tubuli and the loops of Henle. In contrast to these findings the uriniferous tubuli and the malpighian glomeruli did not show any porphyrin fluorescence. It is open to discussion, whether the porphyrin deposits are due to active resorption and secretion or the phenomenon of passive imbition of distinct parts of the renal tubular system is observed.

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Mit Unterstützung des Wissenschaftlichen Fonds der Schering AG, Berlin.     

OH Radical-induced chemical reactions of polynucleotide complexes

Poly(A + U), the 1:1 complex of poly(riboadenylic acid), poly A, and poly(ribouridylic acid), poly U, at pH 8 and self-complexed poly A at pH 4 exist as double helices in dilute aqueous solution. These complexes exhibit a similar behavior as native calf thymus DNA upon irradiation with 16 MeV electron pulses. Thus time resolved Rayleight light scattering measurements showed that crosslinking and double strand breakage can be clearly separated, the former proceeding faster than the latter. The extent to which the two processes occur depends on the ionic strength of the solution. At ionic strenghts exceeding 10 ^?1 mol/l crosslinking is the dominant process indicating that h_crit, the critical length between two single strand breaks for the accomplishment of double strand breaks, is strongly reduced. The investigation of complexes of poly A and Mg²⁺ ions revealed that the destruction of salt bridges is the rate determining process for the decrease of the light scattering intensity due to mainchain scission. This implies that life-times of salt bridges can be determined.