Elektron-Spin-Resonanz-Untersuchungen der Reaktionskinetik strahleninduzierter freier Radikale des Cholesterins

Zusammenfassung Die Reaktionskinetik strahleninduzierter freier Radikale des Cholesterins wurde in flüssiger Phase bei Raumtemperatur mittels ESR-Spektroskopie untersucht. Mit Hilfe eines geeigneten photochemischen Initiationssystems ließen sich in Cyclohexanlösung unter UV-Bestrahlung (235 nm265 nm) genau dieselben freien Radikale des Cholesterins darstellen, die schon früher [9, 7] in röntgenbestrahltem Cholesterinpulver beobachtet worden waren. Bei ausreichendem O₂-Partialdruck (3·10⁴Torr) über der Probenlösung trat das ESR-Spektrum eines Peroxyradikals auf, das mittels der Analyse seiner Reaktionsprodukte (7-Hydroxy-Cholesterin und 7-Keto-Cholesterin) mit dem Cholesteryl-7-peroxyradikal identifiziert wurde. Die Kinetik sowohl der Bildung als auch des Zerfalls des Radikals entsprachen einer Reaktion von 2. Ordnung. Die Geschwindigkeitskonstante für den bimolekularen Zerfall, eine Disproportionierung in Alkohol und Keton unter Abgabe eines Moleküls O₂, wurde bei Raumtemperatur zuk ₂=(1,8 _–0,6 ^+0,9 )·10⁶ sec^–1M^–1·l bestimmt. Ferner wurde gezeigt, daß das Cholesteryl-7-peroxyradikal aus dem freien Radikal Cholesteryl-7 durch Anlagerung eines Moleküls O₂ entsteht. Für die Geschwindigkeitskonstante dieser Reaktion ergab sich eine untere Schranke vonk ₁=0,40·10¹⁰ sec^–1M^–1·l.

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Electron spin resonance investigations on radiation-induced free radicals of cholesterol in liquid phase

Summary The reaction kinetics of radiation-induced free radicals of cholesterol was studied in liquid phase at room temperature by means of e.s.r. spectroscopy on a solution of cholesterol in cyclohexane. Using a convenient photochemical initiation system, just those free radicals of cholesterol could be generated by the filtered u.v. radiation from a Xe high pressure lamp (235 nm265 nm) as were observed already a decade ago by Gordy [9] and by Ehrenberg, Löfroth [7] in X-irradiated cholesterol powder. At sufficiently high O₂-pressures (3·10^–4 Torr) over the sample solution a peroxy radical e.s.r. spectrum arose during u.v. irradiation which was identified by product analysis (7-hydroxy-cholesterol and 7-keto-cholesterol) to be dueto a cholesteryl-7-peroxyradical. The radical'sgeneration and decay kinetics was governed by a second order reaction. The velocity constant for bimolecular decay of the cholesteryl-7-peroxyradical was found to be k₂=(1.8 _–0,6 ^+0,9 )·10⁶sec^–1M^–1·l at room temperature. Furthermore it could be shown that the cholesteryl-7-peroxyradical was built up by the addition of one molecule of O₂ to a cholesteryl-7 free radical. For this reaction a value ofk ₁=0.4·10¹⁰ sec^–1 M^–1·l was estimated as a lower limit of the velocity constant.

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Die Arbeit stellt einen Auszug aus einer Dissertation an der Technischen Hochschule München dar.     

Kurze Mitteilungen

Ohne Zusammenfassung     

Growth and reproduction as a function of temperature and salinity inClava multicornis (Cnidaria,Hydrozoa)

Summary 1. Rates of growth (length increase of stolons) and of asexual reproduction (increase in number of polyps) were determined in secondaryClava multicornis colonies of a clone exposed to 12 different combinations of water temperature and salinity (12°, 17°, 22° C; 16 , 24 , 32 , 40 S). Sexual reproduction (via gonophores) has been observed only at 12° and 17° C; temperature and salinity ranges are narrower for sexual than for asexual reproduction.2. The data obtained are insufficient for a detailed analysis; they provide, however, interesting insights into the variability of growth and reproduction ofC. multicornis caused by different intensities of temperature and salinity.3. It appears that temperature requirements for maximum colony increase are reduced as the colony grows older.4. One feeding period per 24 hours seems insufficient for maximum growth and reproduction at the higher temperature levels, especially at 22° C.5. The different degrees of environmental stress endured during the initial period of transfer into the test combinations of temperature and salinity have affected the resulting colony size at least up to an age of 39 days. More appropriate criteria for assessment of rates of growth and reproduction are therefore the doubling times (number of days within which stolon length and polyp numbers taken 20 days after initiation of experiments have doubled).6. On the basis of doubling time values, increase in stolon length is progressively reduced with increasing water temperature (12°, 17°, 22° C). At 12° and 17° C stolons grow fastest in 32 , followed by 24 , 16 and 40 S; at 22° C stolon growth rates are identical in 32 and 24 S.7. Doubling times of polyp numbers per colony show a less obvious trend. In 56-day-old colonies, however, stolon length and polyp number are modified to similar degrees by the various temperatures and salinities offered. The sequence of temperatures causing fastest increase in polyp number is 12°>17°>22° C; the respective sequence of salinities reads: 24 , 32 , 16 , 40 S.8. Stolon length and polyp number per colony increase exponentially; most curves obtained exhibit undulations indicating endogenous growth rhythms.9. During the initial period of transfer into the final test media, asexual reproduction via budding seems to have been stimulated by a reduction in salinity.10. The doubling times obtained forC. multicornis are considerably longer than those found forCordylophora caspia and indicate that our culture conditions may have been suboptimal.

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Wachstum und Reproduktion als Funktion von Temperatur und Salzgehalt beiClava multicornis (Cnidaria, Hydrozoa)

Kurzfassung Einzelpolypen eines Klons vonC. multicornis Forskål wurden schrittweise in 12 verschiedene Temperatur-Salzgehalts-Kombinationen überführt und — während sie zu neuen Kolonien heranwuchsen — das Längenwachstum ihrer Stolonen, die Geschwindigkeit ihrer asexuellen Vermehrung durch Knospung neuer Hydranthen sowie die Gonophorenausbildung (sexuelle Fortpflanzung) registriert. Die erhaltenen Daten sind unzureichend für eine detaillierte Analyse, gewähren jedoch interessante Einblicke in die Bedeutung der verschiedenen Temperatur- und Salzgehaltsbedingungen für Wachstum und Vermehrung. Die anfängliche, schrittweise Überführung in die Testmedien verursacht per se Leistungsunterschiede, deren Auswirkungen sich mindestens bis zu einem Alter von 39 Tagen verfolgen lassen. Doubling times stellen daher objektivere Kriterien dar als absolute Zuwachswerte. Die doubling times von Kolonien, welche länger als 20 Tage in den Testmedien gewachsen waren, zeigen eine Verringerung der Stolonenzuwachsrate mit steigender Temperatur (12°, 17°, 22° C). Die Reihenfolge der fördernden Wirkung der einzelnen Salzgehaltsstufen ergibt sich zu 32 , 24 , 16 , 40 S. Im Prinzip ähnliche Verhältnisse liegen hinsichtlich der asexuellen Vermehrungsrate vor. Bemessen an den getesteten Kriterien scheinen die Temperaturansprüche mit zunehmendem Koloniealter abzunehmen. Die errechneten doubling times sind wesentlich länger als beiCordylophora; möglicherweise deutet dieser Unterschied auf inadäquate Kulturbedingungen (Fütterung, Wasserbewegung) hin.

     

Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. strain ATCC 33790.

Arthrobacter sp. strain ATCC 33790 was grown with pentachlorophenol (PCP) as the sole source of carbon and energy. Crude extracts, which were prepared by disruption of the bacteria with a French pressure cell, showed no dehalogenating activity with PCP as the substrate. After sucrose density ultracentrifugation of the crude extract at 145,000 x g, various layers were found in the gradient. One yellow layer showed enzymatic conversion of PCP. One chloride ion was released per molecule of PCP. The product of the enzymatic conversion was tetrachlorohydroquinone. NADPH and oxygen were essential for this reaction. EDTA stimulated the enzymatic activity by 67%. The optimum pH for the enzyme activity was 7.5, and the temperature optimum was 25 degrees C. Enzymatic activity was also detected with 2,4,5-trichlorophenol, 2,3,4-trichlorophenol, 2,4,6-trichlorophenol, and 2,3,4,5-tetrachlorophenol as substrates, whereas 3,4,5-trichlorophenol, 2,4-dichlorophenol, 3,4-dichlorophenol, and 4-chlorophenol did not serve as substrates.     

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protistTrichomonas vaginalis

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones fromTrichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which containedT. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of theT. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists likeEntamoeba histolytica, Trypanosoma cruzi, andLeishmania infantum.     

Analysis of DNA polymerase activity in Petunia protoplasts treated with clastogenic agents

Clastogenic agents, i.e. agents that can induce chromosome or DNA breakage, have been shown to enhance the rale of direct gene transfer to protoplasts. The effect was analysed at the enzymatic level using protoplast homogenates as well as intact protoplasts. For that purpose existing procedures were modified to enable measurement of DNA polymerase in vivo. In the system used, external DNA was able to enter the cells without the addition of membrane-permeabilizing compounds. When comparing total DNA polymerase activity of protoplasts irradiated with X-rays or UV-light with that of untreated cells we did not observe significant differences. Incubation of protoplasts with high doses of bleomycin affected total DNA polymerase activity negatively. but dideoxythymidine triphosphate-sensitive activity was not influenced. We conclude that the DNA strand-breaks induced by low doses of X-rays. UV-light or bleomycin do not increase the total or the repair-DNA polymerase activity and. therefore. that the increase in the transformation rates after DNA strand-breaking is not preceded by enhanced DNA polymerase activity.     

Presence and role of jasmonate in apple embryos

(-)Jasmonic acid (JA) was identified in extracts front embryos of apple (Mulus domestica) by combined gas chromatography-mass spectromelry. Quantification of JA in embryos isolated from seeds at different perexts of stratification by gas chromalography combined with mass spectrometry/selective ion monitoring indicated a sharp peak at day 30. At the same time the maximal ratio of conjugated to free JA was found by enzyme-linked imrnunosorbent assay (ELISA). Germination of embryo.s was stimulated by added JA and inhibited by salieylhydroxamic acid (SHAM, an inhibitor of lipoKygenase). Both stimulation and inhibition disappeared in embryos stratified for more than 30 days. Methyl jasmonate was more effective in stimulation of embryo germination than free JA. while JA-isoleucine inhibited germination. The possible mechanism responsible for changes in JA level as wel! as the role of JA and its conjugates in removal of dormancy in apple seeds are discussed.     

Molecular and genetic characterisation of German families with paramyotonia congenita and demonstration of founder effect in the Ravensberg families

Eighteen German families with a history of paramyotonia congenita (PC) were characterised by genetic und mutational analysis at the SCN4A locus, which encodes the -subunit of the adult skeletal muscle sodium channel. We concentrated our analysis primarily on these families to test the hypothesis that a predominance of one common mutation occurs in all German PC families and that this mutation arose in a common ancestor originating in the North-West of the country. The present eighteen PC families exhibit two different mutations (R1448C and R1448H) on various SCN4A dinucleotide repeat haplotypes and therefore the majority of the mutations probably occurred independently. However, the R1448H mutation is extremely frequent in the North-West of Germany (Ravensberger Land) on a specific SCN4A microsatellite haplotype, indicating a founder effect within this subpopulation. Our results suggest that the R1448C/R1448H mutations are by far the most common to be associated with the PC phenotype in the German population.This paper is dedicated to Professor Dr. Dr. Peter Emil Becker on the occasion of this 85th birthday