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.     

Ultrastructure des cellules germinales au cours du développement embryonnaire du Lézard vivipare (Lacerta vivipara Jacquin)

Résumé Les gonocytes primaires sont relativement pauvres en polysomes et l'ergastoplasme granulaire est très réduit. Le reticulum endoplasmique de type lisse se développe au cours du développement embryonnaire. L'appareil de Golgi est bien représenté et localisé au niveau de la calotte juxtanucléaire mitochondriale. Les liposomes cytoplasmiques sont très nombreux.Cette étude précise la structure du nucléole «annulaire» et de la «masse paranucléolaire» observés en microscopic photonique. Des modifications nucléolaires sont constatées au cours du développement embryonnaire. Certains aspects ultrastructuraux sont vraisemblablement en rapport avec le déplacement autonome des gonocytes. La signification physiologique du nucléole «annulaire» et de la «masse paranucléolaire» est envisagée.

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Ultrastructural study on the primordial germ cells during embryonic development of Lacerta vivipara Jacquin

Summary The primordial germ cells of Lacerta vivipara have relatively few free polysomes and little ergastoplasm. Smooth endoplasmic reticulum increases during embryonic development. The Golgi apparatus is well developed and lies close to the mitochondrial juxtanuclear cap. This study shows the ultrastructure of the ring-shaped nucleoli and the masse paranucléolaire. Modifications in nucleolar structure are observed during embryonic development. Some ultrastructural features are probably related to the ameboid movement of the primordial germ cells. The physiological meaning of the ring-shaped nucleoli and the masse paranucléolaire is considered.

Avec la collaboration technique de Mme. M. Hubert.     

Die auswirkungen der körpergrösse von Aphelinus semiflavus howard (HYM., Aphelinidae) auf einige Organe und ihre leistungsfähigkeit

Zusammenfassung Die Körpergröße von Aphelinus semillavus How. variiert in Abhängigkeit von Große und Qualität des Wirtes. Tiere aus dem gesamten Variationsbereich wurden auf größenabhängige Proportionsverschiebungen untersucht.Relative Länge und Breite des Kopfes (bezogen auf die Körperlange) nehmen mit zunehmender Körperlange ab. Die relative Lange des Abdomens wächst etwa um den gleichen Betrag:Es besteht eine negative Korrelation zwischen der relativen Länge des letzten Fühlergliedes, des Trägers wichtiger Sinnesorgane, und der Körperlange. Seine Oberfläche steht in einem etwa konstanten Verhältnis zur Körperlänge. Die Anzahl der Porenplatten nimmt mit der Körperlänge nur wenig, ihre Länge deutlicher zu.Im Komplexauge wächst die Anzahl der Ommatidien etwa proportional zur Korperlange, während seine gesamte Fläche zwar linear, aber ïiberproportional ansteigt, was durch zunehmende Große der einzelnen Facetten erreicht wird.Die Länge der Vorderfliigel ist bei Männchen und Weibchen von mehr als 1 mm Länge etwa der Körperlange proportional. Unterhalb 1 mm nimmt die relative Flügellänge mit der Körperlange ab, bis die Flügel nur noch funktionslose Rudimente bilden. Die Relation Flügel-fläche:Körpergewicht ist bei mittelgroßen Tieren am günstigsten.Jedes Ovar hat stets drei Ovariolen, unabhängig von der Körperlänge. Große Weibchen legen nur etwas größere, aber bedeutend mehr Eier als kleine. Die Anzahl der (am 7.–10. Lebenstag) täglich abgelegten Eier steigt etwa linear mit dem Logarithmus des Körpergewichts an.Die Eiablage wird während des ganzen Lebens (im Labor etwa 40 Tage) fortgesetzt, ist aber zum. Ende hin etwas geringer. Relativ zum Körper-gewicht produzieren mittelgroße Weibchen die größte Eimasse. Die Anzahl der zum Nahrungsgewinn ausgesaugten Läuse steigt mit dem Körpergewicht.Zwischen der relativen Länge des Legestachels und der Körperlange besteht eine fast lineare negative Korrelation.Die gröBenabhangigen Proportionsverschiebungen werden als Auswirkungen unterschiedlicher Wachstumstendenzen der einzelnen Organe gedeutet, die ihrerseits genetisch fixiert sind und den biologischen Sinn haben, die Lebensf ähigkeit modifikatorisch kleiner Exemplare zu sichern und damit die Ausnutzung entsprechend kleiner Wirte zu ermöglichen.Die Bedeutung der Proportionsverschiebungen für die Leistung der ganzen Population wird diskutiert. Sic ist abhängig von der Häufigkeits-verteilung der verschiedenen Körpergrößen und these wiederum von der Art-Zusammensetzung und Ernährung der Wirtsfauna.

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Summary The body size of Aphelinus semiflavus How. varies with the size and the quality of the hosts. Specimens from the whole range of variation were investigated for differences between organ proportions, which depended on size.The relative length and width of the head (based on the body length) are reduced with increasing body length. The relative length of the abdomen increases approximately by the same amount.There is a negative correlation between the relative length of the last joint of the antennae and the body length. Its surface shows a nearly constant proportion to the body length. The length of the pore plates increases with the body length more distinctly than their numbers.Within the compound eye, the number of ommatidia increases almost proportionally to the body length; the whole area of the eye increases more than proportionally because the size of the single facets also becomes greater.When the body length is greater than 1 mm, the length of the forewings is nearly proportional to the body length. At less than 1 mm, the relative length of the fore-wings is reduced with declining body length until, finally, only rudiments without any function remain. The ratio between wing area and body weight is most favourable at medium body size.Three ovarioles, independent of the body size, are present in each ovary. Big females produce considerably more eggs than small ones. The size of their eggs is only slightly enlarged. The daily egg production (at the 7^th–10^th day of life) increases nearly linearly with the logarithm of body weight. Oviposition is continued throughout the life-time of the insect (ca. 40 days in the laboratory) but it is somewhat reduced at the end. Relative to their body weight, females of medium size produce the biggest egg masses. The number of aphids utilized by the parasite for nourishment increases with the body weight of the female.A negative correlation exists between the relative length of the extended ovipositor and the body length.The proportion differences of the organs, dependent on size, are interpreted to be consequences of different growth tendencies which are genetically fixed. Apparently, the biological role of such different growth tendencies is to ensure the viability of phenotypically small specimens and in this way to make the utilisation of small hosts possible.The importance of the differences of body proportions for the effect of the whole population is discussed. It depends on the frequency distribution of the different body lengths which itself is the consequence of the composition of host species and the nutriation of the hosts.

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Herrn Prof. Dr. Hans Braun, emer. Direktor des Instituts für Pfanzenkrankheiten an der Universität Bonn, zu seinem 70. Geburts'ag am 2. Februar 1966 gewidmet.     

Direct effects of serotonin and ketanserin on the functional morphology of embryonic chick skin in vitro

Summary Different organotypical culture methods are used to test the direct effects of serotonin and ketanserin, a S₂, α₁, and H₁ receptor antagonist in vascular tissue, on fibroblasts and epidermal cells of embryonic chick skin in vitro. From light microscopic and electron microscopic analyses, we learn that serotonin enhances keratinization and differentiation, whereas ketanserin reduces differentiation in comparison to the control cultures. Incorporation data of fragments cultured with [³H]thymidine show that ketanserin, within a dose range from 0.05 to 5 μg/ml, stimulates proliferation. Serotonin at a concentration of 10 μg/ml slightly slows down proliferation, whereas lower doses of 0.1 and 1 μg/ml result in tritium activities that do not differ from control cultures. This investigation was financially supported by the National Fund of Scientific Research, Belgium, 3.0022.87.     

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

Summary In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven⁸⁶Rb⁺ fluxes through K⁺ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10^–7 m free Ca²⁺. This suggests an important role of cytoplasmic Ca²⁺ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K⁺ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca²⁺-activated K⁺ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H⁺ and tetraethylammonium show that both high-and low-sensitive⁸⁶Rb⁺ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.     

Pharmacological Characterization of Somatostatin Receptors in the Rat Cerebellum During Development

Abstract: Somatostatin (SRIF) receptors (SRIF-Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF-Rs coincides with the expression of SRIF-like immunoreactivity in the cerebellum. However, the cellular location of SRIF-Rs does not overlap with the distribution of SRIF-like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF-Rs in the immature (13–day-old) rat cerebellum was conducted by means of binding experiments in membraneenriched preparations and autoradiography, using two radioligands, [¹²⁵I-Tyr⁰,D-Trp⁸]SRIF-14 ([¹²⁵I-Tyr⁰,d -Trp⁸]S14) and ^I25I-SMS 204–090. The pharmacological profile of cerebellar SRIF-Rs was compared with that of adult cortical SRIF-Rs. Saturation studies performed in 13–day-old rat cerebellum showed that the A'_D values for [¹²⁵I-Tyr⁰,D-Trp⁸]S14 and ¹²⁵I-SMS 204–090 binding were 0.35 ± 0.04 and 0.39 ± 0.01 nM, respectively. The corresponding B_max values were 52.7 ± 4.8 and 49.9 ± 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high-affinity sites (SSI). Competition studies showed that different D-Trp-sub-stituted analogs displaced [¹²⁵I-Tyr⁰,d -Trp⁸]S14 binding with Hill coefficients >1, a finding indicating the existence of different subtypes of binding sites. When [Tyr⁰,d -Trp⁸]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13–day-old cerebellum and adult cerebral cortex. The higher-affinity sites correspond to the SSI subtype identified in saturation experiments, whereas the lower-affinity sites most likely correspond to the SS2 subtype. Ionic supplementation studies showed that divalent cations were required to obtain maximal specific binding on the SSI sites. In particular, Mn²⁺ was the most efficient cation for promoting binding of [¹²⁵I-Tyr⁰,d -Trp⁸]S14. Addition of GTP to the incubation buffer induced a marked reduction of specific binding. The results obtained by membrane binding assays were similar to those obtained by quantitative autoradiography, a result indicating that the microenvironment of SRIF-Rs was preserved in both types of tissue preparations. Receptors expressed in the developing rat cerebellum exhibited the same K_D and similar pharmacological profile as those observed in the adult rat cortex. These results show that SRIF-binding sites transiently expressed in the external granule cell layer of the cerebellum of young rats are indistinguishable from adult rat brain SRIF-Rs. The extremely high density of SRIF-Rs found in the external granule cell layer in 13–day-old rats suggests that SRIF may play a pivotal role in the proliferation and/or differentiation of these germinative cells.     

Linkage analysis of the Duffy blood group marker with several chromosome 1 genes in an extended pedigree with Charcot-Marie-Tooth disease

Summary We have recently demonstrated tight linkage of the Duffy blood group marker to the -spectrin gene in an extended pedigree with Charcot-Marie-Tooth neuropathy. To determine a more precise location of the Duffy blood group locus on the chromosome 1 map we have tested several more chromosome 1 genes for linkage with this marker. We found suggestive linkage with the antithrombin III and apolipoprotein A2 genes and conclusive linkage with the gene coding for -nerve growth factor.     

The roles of calcium and manganese ions in the in vitro conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene by lentil root membranes

Ca²⁺ and Mn²⁺ activate the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) by root microsomes of Vicia lens as they do in other similar systems. The preparation of microsomes in the presence of Mn²⁺ greatly increases their ability to convert ACC into ethylene, without addition of Mn²⁺ in the reaction mixture. Ca²⁺ does not have this property. The effect could not be attributed to Mn²⁺ entrapping into membrane vesicles (sonication followed by repelleting had no effect) but, possibly, in part to Mn²⁺-mediated binding to microsomes of a soluble factor favouring the conversion of ACC to C₂H₄. Although no direct correlation could be established in vitro between ethylene-forming-enzyme (EFE) and peroxidase activities, some soluble peroxidases might be this soluble factor. Mn²⁺ favoured attachment to membranes of some peroxidase activity from the soluble fraction and from commercial HRP and lipoxygenase. This binding effect of Mn₂₊ cannot be readily distinguished from its role in the generation of a chain of free radicals and in redox mechanisms.