Der Stoffwechsel von Indolderivaten in Sinapis Alba L.

Zusammenfassung Die Aufnahme von D,L-Tryptophan in isolierte Hypokotylsegmente von Sinapis alba ist ein aktiver Prozeß; er erfolgt auch gegen ein Konzentrationsgefälle und wird durch 2,4-Dinitrophenol gehemmt. Die Aufnahmerate der D-Isomeren beträgt nur 50% der L-Verbindung.Einzig nachweisbare Umsetzungsprodukte des L-Tryptophans in Sinapishypokotylen waren die Glucosinolate (Senfölglucoside) Glucobrassicin und Neoglucobrassicin. Indolauxine wie IES und IAN traten auch nach Fütterung von radioaktivem Tryptophan in chemisch oder radioaktiv nachweisbaren Mengen nicht auf. Frühere Nachweise dieser Verbindungen beruhen auf Artefaktbildung im Verlauf der Extraktion und Aufarbeitung.Das Endprodukt der Umsetzung von D-Tryptophan ist auch in Sinapis D-Malonyltryptophan.Der Zeitverlauf der Umsetzung von C¹⁴-D,L-Tryptophan und S³⁵-Sulfat in Sinapis wird beschrieben. Ein rascher Einbau der S³⁵-Aktivität in den Thioäther des Glucosinolats beweist eine nennenswerte Reduktion des Sulfats auch im Dunkeln.Zwischen papierchromatographisch und hochspannungselektrophoretisch isoliertem Glucobrassicin und seinem kristallinen Tetramethylammoniumsalz besteht keine Übereinstimmung in der Spaltbarkeit durch Myrosinase.Die Bedeutung der Indolglucosinolate für den Auxinhaushalt und die derzeitige Situation in der Erforschung der Indolauxine in Cruciferen wird diskutiert.

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Metabolism of indole derivatives in Sinapis alba L.II. Investigation of the biogenesis and metabolism of indole glueosinolates with the aid of Ring-Labelled C¹⁴-Tryptophane and S³⁵-Sulphate

Summary D,L-tryptophane is taken up by isolated segments of the hypocotyls of Sinapis alba against a concentration gradient. This process is inhibited by 2,4-dinitrophenol. Both observations suggest an active uptake process. The rate of absorption of the D-isomer is only 50% of that of the L-compound.The glucosinolates (mustard oil glucosides), glucobrassicin and neoglucobrassicin are the only metabolic products of L-tryptophane which we have been able to detect in hypocotyls of Sinapis. Neither by chemical methods nor with the aid of tracer techniques were we able to detect any indole auxin like IAA or IAN. We attribute earlier reports of the occurrence of compounds of this type in Cruciferae to artefact formation in the course of extraction and preparation.The metabolic end product of D-tryptophane in Sinapis is D-malonyl tryptophane.The kinetics of the metabolism of C¹⁴-D,L-tryptophane and S³⁵-sulphate in Sinapsis hypocotyls has been determined. The fast labelling by S³⁵ of the thioether group of glucosinolates demonstrates that sulphate reduction is possible even in the dark.Glucobrassicin isolated by paper chromatography or high voltage electrophoresis differs from its crystalline tetramethylammonium salt in respect to the rate at which it is split by myrosinase.The importance of the indole glucosinolates for the auxin metabolism and the present situation in the investigation of indole auxins in Cruciferae is discussed.

     

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.     

Advanced fertility diagnosis in stallion semen using transmission electron microscopy

Routine semen analysis of stallions is based on light microscopy (LM). However, there are still a number of animals that are subfertile or even infertile not being identified with conventional semen analysis. The objective of this study was to investigate the suitability of transmission electron microscopy (TEM) for advanced fertility diagnosis in stallion. We examined ejaculates of 46 stallions with known fertility. Animals were divided into three different groups: group 1, fertile stallions (pregnant mares> or =70%, n=29); group 2, subfertile stallions (pregnant mares 10-69%, n=14); group 3, infertile stallions (pregnant mares<10%, n=3). Ejaculates were collected in spring 2002. Conventional semen analysis (volume, sperm concentration, motility, live:dead ratio and percentage of morphologically normal sperm) was immediately performed after semen collection. Ultrastructural analysis included the evaluation of 200 acrosomes, heads, midpieces and cross-sections of tails as well as 100 longitudinal sections of tails from every ejaculate. Using LM, we found a significant increase of morphological deviations from 24.5% (x ) in group 1 to 34.5% in group 2 and 73.5% in group 3. Using TEM, we found a significant increase of detached acrosomes from 6.1% in group 1 to 7.6% in group 2 and 21.4% in group 3. Deviations in tubule pattern were also increased (but not significant) from 2.7% in fertile and 2.8% in subfertile to 11.4% in infertile stallions as well as multiple tails from 1.9% in fertile to 2.0% in subfertile and 8.9% in infertile. Our data indicate that TEM is suitable for advanced fertility diagnostic in stallions, giving a connection between fertility and morphology. It suggests that the most likely reason for sub- and infertility in stallion in case of increased LM pathomorphology of semen are acrosomal alterations, especially detached acrosomes.