Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene

Summary A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial -glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of -glucuronidase activity.     

A novel granular cell type of locust Malpighian tubules: ultrastructural and immunocytochemical study

Summary A novel secretory cell type in the initial segment of the Malpighian tubules of the locusts Schistocerca gregaria and Locusta migratoria is described ultrastructurally and studied by means of immunocytochemical techniques. The cells show abundant rough endoplasmic reticulum with interspersed Golgi zones. The richness of the cell secretory machinery and the presence of apical dense plemorphic granules suggest a role in secretion of proteinaceous material to the tubule lumen. The surprising finding of ACTH (1–24)-, -MSH-, and 7B2-like immunoreactivity for this cell is discussed.     

Oxygen transfer properties of bubbles in animal cell culture media

Oxygen transfer rates were determined in a bubble aerated animal cell bioreactor. It was found that the oxygen transfer rates increased in the following order: large bubbles ( approximately 5 mm diameter) < intermediate bubbles ( approximately 1 mm diameter) < micron-sized bubbles ( approximately 100 mum diameter). Under certain conditions, the micron-sized bubbles were capable of achieving oxygen transfer rate up to 100 h(-1), a 10-20-fold higher transfer rate than the large bubbles. The effects of medium composition on oxygen transfer rates were different for the three ranges of bubbles studied. For the large bubbles, oxygen transfer rates decreased with increasing medium complexity. The lowest oxygen transfer rate was found in new-born calf serum (NBCS) and/or Pluronic F-68 supplemented media. For the intermediate and micron-sized bubbles, supplementation with NBCS into the culture media resulted in decreased oxygen transfer rate. However, further supplementation with Pluronic F-68 enhanced oxygen transfer rate greatly for both types of bubbles. The highest oxygen transfer rate was found for micron-sized bubbles in Pluronic F-68 supplemented media containing antifoam agent and NBCS.     

Triterpenes of Prunus serotina and P. lusitanica

The leaves of Prunus serotina and P. lusitanica contain a new triterpene, 2α,3α-dihydroxyurs-12-en-28-oic acid, isolated in form of its methyl ester. Other triterpenes present in these species are ursolic acid and ursol aldehyde. P. lusitanica also yields friedeline.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

-Isopropylmalate (IPM) synthase, the first enzyme in the biosynthesis of l-leucine, was purified to a specific activity of 12 mole/min x mg protein from the valine-isoleucine double auxotrophic mutant A-81 of the hydrogen bacterium Alcaligenes eutrophus H 16. The activity in crude extracts of derepressed cells was 0.106 moles of isopropylmalate formed per min and per mg protein. Gel electrophoresis and regel electrophoresis of the isolated main band resulted in several distinct bands, which were not altered by the additions of substrate -ketoisovalerate, feedback inhibitor leucine or other effectors.The isoelectric points of the enzyme protein was between 3.9 and 4.0. The molecular weight was 114500 daltons and 100000 respectively in the absence and presence of the feedback inhibitor leucine. The enzyme activity depended strongly on the pH, the optimum is at pH 8.2. The enzyme was could labile and exhibits temperature anomalies.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Eusynaptomyces benjaminii,spec. nova, (Ascomycetes,Laboulbeniales) und seine Anpassungen an das Fortpflanzungsverhalten seines WirtesEnochrus testaceus (Coleoptera,Hydrophilidae)

Eusynaptomyces benjaminii is described as a new species of the ectoparasiticLaboulbeniales (Ascomycetes). It exists only on two very restricted areas of the body (= position specifity) of its hostEnochrus testaceus (F.) (Coleoptera, Hydrophilidae): on the claws of the right fore-leg and on the lower side of the frontal border of the pronotum. In these two habitatsEu. benjaminii develops two extremely different growth-forms. Male and female hosts are parasitized on somewhat different parts of their body. This can be explained by their mating behaviour. The growth-forms ofEu. benjaminii are so different that one ignorant of the biology of hosts and parasites, might regard them as members of different species or even genera. They are to be interpreted as adaptations of one species to growth positions and mating behaviour of the host. There is no sex-of-host specifity as assumed by certain authors for several species of theLaboulbeniales.

     

Mutagenic activation of biliary metabolites of 1-nitropyrene by intestinal microflora

To investigate the modifying role of intestinal microflora in the metabolism of chemical carcinogens in vivo, we subjected bile from Fischer rats treated per os with chemical carcinogens and related compounds to a mutagenicity assay in the presence and absence of a cell-free extract from human feces. A mixture of the bile sample and potassium phosphate buffer was incubated in the presence or absence of human cell-free fecal extract and then further incubated with a bacterial suspension of Salmonella typhimurium tester strains TA98 or TA100. Bile from rats treated with 1-nitropyrene (1-NP) produced about 2700 and 400 revertants per plate in strain TA98 in the presence and absence of the fecal extract, respectively. There was a drug dose- and bile volume-related response. Treatment of 1-NP-bile with beta-glucuronidase, but not aryl sulfatase, enhanced its mutagenicity. Cell-free extracts of some strains of intestinal bacteria (Bacteroides fragilis ATCC 12044, B. vulgatus ATCC 8482, B. thetaiotaomicron ATCC 12290, Bacteroides sp. strain 524, Eubacterium eligens VPI C15-48, Peptostreptococcus sp. strain 204 and Escherichia coli A-5-18) also enhanced the mutagenicity of 1-NP-bile. These bacterial cell-free extracts hydrolyzed the synthetic beta-D-glucuronides of phenolphthalein and/or p-nitrophenol. These data indicate that the glucuronide(s) of 1-NP-metabolite(s) secreted into bile can be hydrolyzed in the intestine by bacterial beta-glucuronidases to potent mutagenic aglycone(s).     

Ethanol directly increases dihydrotestosterone conversion primarily to 5 alpha-androstan-3 beta, 17 beta-diol in rat Leydig cells

Our studies demonstrate that direct stimulation of dihydrotestosterone metabolism by ethanol (2.2 - 65 mM) in rat Leydig cells primarily involves an increase in 5 alpha-androstan-3 beta, 17 beta-diol. Although the enzyme catalyzing this conversion, 5 alpha-androstane-3 beta-hydroxysteroid dehydrogenase, is localized in the microsomal fraction of Leydig cells, ethanol does not increase 5 alpha-androstan-3 beta, 17 beta-diol formation in isolated microsomes, presumably because of the removal of soluble alcohol dehydrogenase activity, which we propose mediates this action. Because 5 alpha-androstan-3 beta, 17 beta-diol is generally considered a weak or inactive androgen, this effect may function to decrease dihydrotestosterone secretion by Leydig cells and/or to reduce the availability of this androgen in responsive tissues.     

Cyanogenic compounds as protecting agents for organisms

Biochemical and physiological arguments and several plant-predator relationships described in the literature are presented in which cyanogenesis plays a role as a protecting process. HCN arising from the cleavage of cyanogenics is regarded to be the most important agent, but also the cyanogenic itself, carbonyls and -cyanoalanine, which are products of degradation processes of cyanogenics, may possess protecting properties. Some examples show that these substances are also utilized by arthropods. This presents the opportunity to look at a coevolutionary system combined of snails, plants, moths and moth-parasites in which cyanogenesis obviously plays an interesting role.Lecture presented during the Tagung der Deutschen Botanischen Gesellschaft, Vienna, September 1984.     

Thin-layer isoelectric focusing of polyphenoloxidase of Sephadex and its detection by the print technique.

Separation of polyphenoloxidase isoenzymes based on their charge properties was achieved by isoelectric focusing on Sephadex G-75 thin layers containing a mixture of ampholytes in the pH ranges 4–6 and 3–10. The separated isoenzymes can be detected as colored zones by a print technique in which a dried filter paper, previously buffered with 0.1 m phosphate buffer, pH 7,0, and impregnated with 1% substrate in methanol, is placed onto the gel layer. d-Catechin and tyramine were the best substrates for detecting the diphenolase and monophenolase activities, respectively. Using this technique, two commercial preparations of mushroom tyrosinase were found to consist of 7 and 15 isoenzymes, while enzyme preparations from two potato varleties showed 11 to 15 isoenzymes. The isoenzymes of potato and mushroom polyphenoloxidase showed marked differences in their pI values.