Purification of antibacterial attacins induced in Hyalophora cecropia pupae immunized with Enterobacter cloacae C7-501 using ion-exchange chromatography,hydrophobic interaction chromatography,and Rotofor® isoelectric focusing

Hyalophora cecropia pupae were infected by Enterobacter cloacae C7-501 to induce antibacterial attacins for purification. The induction of attacins in immunized pupae was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), and Rotofor® isoelectric focusing (ISEF) were applied to isolate attacins from the hemolymph. IEC separated attacins from most hemolymph proteins, but the fractions containing attacins also had other proteins of 20 and 64 kDa in length. In IEC, attacin was eluted with ~0.2 M NaCl. The best conditions for IEC were pH 9, flow rate of 2 mL/min, with step elution (0.025, 0.05, 0.075, 0.1, 0.2, 0.4 and 1.0 M NaCl). In HIC, most other proteins were eluted with the ammonium persulfate treatment. HIC isolated attacin proteins under hydrophobic conditions, at ~50% EtOH. However, the fraction with attacins also contained other proteins. The Rotofor® ISEF produced fractions containing attacins at isoelectric points ranging between 5.7 and 8.3. However, non-specific proteins were detected in the fraction samples, and the recovery of attacins was low. The purification efficiency of ISEF was lower than IEC and HIC. In this study, the expression of attacins was induced in H. cecropia pupae infected with E. cloacae C7-501, and attacins could be purified by IEC and ISEF. Overall, IEC provided better separation of attacins from the hemolymph of H. cecropia pupae immunized with E. cloacae bacteria than HIC and Rotofor® ISEF.     

Über die chromotropen Zellen der Nebenniere vom Wasserfrosch

Zusammenfassung Aus dieser neuerlichen Untersuchung der zuerst von Stilling beschriebenen Sommerzellen ergibt sich somit als einzige Übereinstimmung in den sonst widerspruchsvollen Angaben des Schrifttums, daß sie in ihrem Vorkommen nicht auf den Sommer beschränkt sind, weshalb sie von mir nach ihrer färberischen Eigentümlichkeit als chromotrope Zellen bezeichnet werden. Da ihr isoelektrischer Punkt bei etwa p_H 5 liegt, verhalten sie sich gegenüber Farbstoffen nicht ausschließlich (acido-) oxyphil, doch sind sie auch keine Mastzellen oder überhaupt während der Entwicklung veränderte Wanderzellen, sondern eine besondere Art autochthon entstandener Nebennieren-Epithelzellen, deren Körnchen saure Polysaccharide enthalten. Ihre funktionelle Bedeutung muß erst geklärt werden. Dabei ist es besonders bemerkenswert, daß sich das Vorkommen dieser Zellen auf Rana esculenta und eine Abart von ihr sowie exotische Verwandte beschränkt, während sie bei Rana temporaria und deren nächsten Verwandten immer fehlen. Die gegenteiligen Angaben der Literatur beruhen wahrscheinlich teilweise auf unzutreffender Bestimmung der Art und im übrigen wohl auf Verwechslung mit gekörnten Wanderzellen, was besonders bei der Entwicklung zu falschen Vorstellungen führen kann.Herrn Professor Alfred Kohn in dankbarer Erinnerung gewidmet.     

Expression and Promoter Activity of Genes for Isozymes of Horseradish Peroxidase

The expression and promoter activity of genes for isozymes ofhorseradish peroxidase, namely, prxCla, prxClb, prxC2 and prxC3,were studied. Organ-specific expression of these genes in horseradishplants was examined by Northern blot analysis. The group ofprxCl genes was expressed mostly in stems, while prxC2 and prxC3were expressed to a greater extent in roots. Hardly any expressionof any of the genes was detected in leaves. In transient-expressionassays with tobacco protoplasts, about 500 bp of the 5'-noncodingregions of each of the genes, ligated to the gene for ß-glucuronidase(GUS), exhibited significant promoter activity. In particular,the fragments extending from the initiation codon of the prxC2gene to –529 bp and –1 kbp supported high levelsof GUS activity, which were 4.4 and 11.4 times respectively,the activity observed under control of the 35S promoter fromcauliflower mosaic virus (CaMV). Conserved enhancer sequencesof human genes were found in the 5'-flanking region of prxC2,and deletion of the regions that contained the enhancer sequencesreduced the GUS activity. High levels of GUS activity were observedin transgenic tobacco plants that contained 1 kbp of the 5'flanking region of prxC2 fused to the GUS gene. GUS activitywas diminished when deletion from the 5' end extended as faras the CAAT box. No significant organ-specific expression ofGUS was observed with any such deletion. (Received April 15, 1992; Accepted September 11, 1992)     

Nature of Enhanced Severity of Anthurium Root Rot by Diuron Treatment for Weed Control

Diuron treatment for weed control greatly increased anthurium root rot caused by Pythium splendens, P. spinosum, P. vexans and Calonectria crotalariac. Diuron in agar medium was inhibitory to the growth of mycelium, formation and germination of sporangia of P. splendens. Sporangia of P. splendens produced in diuron-amended medium did not differ in pathogenecity to anthurium roots from those produced in diuron-free medium. When diuron was applied to kill weeds in the planting medium, the population of P. splendens in it was not decreased during the test. Diuron was inhibitory to a number of micro-organisms in the platiting medium. Exudation of anthursum roots was not increased by diuron treatment. Increase in severity of anthurium root rot by diuron treatment was similar whether the experiments were performed in the presence or absence of planting medium, suggesting that the enhancing effect of diuron on root rot is mainly due to an increase in susceptibility of the host plants.     

IN VIVO EFFECTS OF CARDIOTROPHIN-1

Cardiotrophin-1 (CT-1) is a recently discovered cytokine that was isolated based on its ability to induce cardiac myocyte hypertrophy in vitro. In this study, the effects of chronic administration of CT-1 to mice (0.5 or 2 μg by intraperitoneal injection, twice a day for 14 days) were determined. A dose-dependent increase in both the heart weight and ventricular weight to body ratios was observed in the treated groups. The body weights of the animals were unaffected. These results indicate that CT-1 can induce cardiac hypertrophy in vivo. CT-1 was not specific for the heart, however. It stimulated the growth of the liver, kidney, and spleen, and caused atrophy of the thymus. CT-1 administration also increased the platelet counts by 70%, with no change in mean platelet volume. Red blood cell counts were increased in the treated animals, and there was a concomitant increase in haemoglobin concentration. Thus, CT-1 has a broad spectrum of biological activities in vivo. This observation is consistent with previous in-vitro findings showing that the mRNA for CT-1 is expressed in several tissues, and that CT-1 can function through binding to the leukaemia inhibitory factor (LIF) receptor and signalling through the gp130 pathway.     

Sensitivity of random amplified polymorphic DNA analysis to detect genetic change in sugarcane during tissue culture

Random amplified polymorphic DNA (RAPD) analysis using 10-mer oligonucleotide primers efficiently differentiated sugarcane cultivars and proved suitable for detecting gross genetic change such as that which can occur in sugarcane subjected to prolonged tissue culture, for example in protoplast-derived callus. However, RAPD analysis was not sufficiently sensitive to detect smaller genetic changes that occur during sugarcane genetic transformation. The length of DNA scored for polymorphism per primer averaged 13.2 kb, or 0.0001% of the typical sugarcane genome size of 1.2 × 10₇ kb (2C). RAPD analysis of sugarcane plants regenerated from embryogenic callus revealed very few polymorphisms, indicating that gross genetic change is infrequent during this tissue culture procedure, although epigenetic effects result in transient morphological changes in regenerated plants. More sensitive variations on the RAPD technique may increase the practicality of DNA-based screening of regenerated plant lines to reveal somaclonal variants.     

Temperature and dissolved oxygen concentration as parameters of Azotobacter chroococcum cultivation for use in biofertilizers

Summary Azotobacter chroococcum was grown in continuous culture at two temperatures (30 °C and 20 °C) and different dissolved oxygen tensions (DOT) (30 % to 40 % and 70 % to 80 % of air saturation), respectively. At the temperature of 30 °C and low DOT a relatively high volumetric productivity and efficiency of nitrogen fixation were obtained. After lowering the temperature to 20 °C, an intensive formation of cysts was observed associated with a drastic decrease of the bacterial growth. Bacteria in the form of cysts kept their physiological activity for a long period of time depending on temperature and preparation.