Establishment of transformed root cultures of Perezia cuernavacana producing the sesquiterpene quinone perezone

Summary The sesquiterpene quinone currently known as perezone is abundantly produced by the roots of Perezia cuernavacana. This compound is of biotechnological interest since it may be used as a pigment and has several pharmacological properties. In this work we demonstrate that perezone is also produced in transformed root cultures of P. cuernavacana. Hairy roots were induced by inoculation of internodal segments of sterile plants of P. cuernavacana with Agrobacterium rhizogenes AR12 strain. The axenic liquid MS medium cultures of the hairy roots isolated from the internodes showed active growth in the absence of growth regulators. The transformed nature of the tissue was confirmed by genomic integration (PCR and slot blot hybridization) and expression (enzyme activity) of the marker gus-gene. The production of perezone by a transformed root culture was evidenced by IR spectroscopy. Our results offer an alternative for enhanced production of perezone and represent an advantage over its extraction from natural plant populations which present problems in their agronomic culture.     

Spermatophore development and sperm ultrastructure inCraterostigmus tasmanianus (Chilopoda,Craterostigmomorpha)

 Spermatophore development and ultrastructure of the mature sperm of Craterostigmus tasmanianus were studied using light and electron microscopy. In C. tasmanianus, as in the Scolopendromorpha, the spermatophore develops within the vas deferens. The latter consists of three parts, each with a different morphology. The first may be involved in guiding the sperm to roll up into typical ring-like structures, while the other two, which show an evident secretory activity, secrete the acellular wall of the spermatophores. The ultrastructure of mature spermatozoa showed that a very close similarity exists between Craterostigmomorpha and Lithobiomorpha, especially regarding the organization of the connecting piece. Based on this similarity, we consider the Craterostigmomorpha together with the Scolopendromorpha, Geophilomorpha and Lithobiomorpha (=Pleurostigmophora) to be the sister group of the Scutigeromorpha. Accepted: 2 June 1996     

Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use in purifying the minor X24 xylanase

 Using recombinant DNA techniques, an Aspergillus nidulans multicopy transformant for the gene xlnB coding for the minor X₂₄ xylanase has been constructed. When grown on glucose as sole carbon source this transformant secretes 114 U of xylanase (mg protein)^-1. In this culture condition, X₂₄ is the only xylanase secreted and the predominant protein in the culture filtrate. This strategy has been used to purify the X₂₄ enzyme to homogeneity. The purified xylanase showed a single band on sodium dodecyl sulphate/ polyacrylamide gel electrophoresis with a molecular mass of 24 kDa and had an isoelectric point of approximately 3.5. The enzyme was a non-debranching endo-1,4-β-xylan xylanohydrolase highly specific for xylans and showed optimal activity at pH 5.5 and 52°C. The X₂₄ xylanase had a Michaelis constant, K _m, of 12.43 mg oat spelt xylan ml^-1 and a V _max of 1639 μmol min^-1 (mg protein)^-1. Received: 17 May 1995/Received last revision: 25 September 1995/Accepted: 29 September 1995     

Effect of oleuropein and sodium chloride on viability and metabolism of Lactobacillus plantarum

 The effect of the addition of oleuropein (OLP) and NaCl on the growth and the DL-lactic acid production of Lactobacillus plantarum DSM 10492 has been investigated by using an unconventional medium. The growth of L. plantarum was not inhibited by the addition of increasing amounts of untreated OLP in the presence or absence of glucose. However, bacterial cells grew in quantity slightly with OLP alone. The increased addition of NaCl was associated with a delay in growth. Moreover, there was no growth with 8% NaCl. The addition of both NaCl and OLP resulted in growth inhibition, and the survival of cells decreased strongly. The main fermentation product was DL-lactic acid, but acetic acid was also detected after a prolonged incubation. L. plantarum produced DL-lactic acid in the presence of OLP alone but its formation decreased with increasing levels of OLP. On the other hand, heat-treated OLP had a bactericidal effect. Received: 16 October 1995/Received last revision: 5 February 1996/Accepted: 12 February 1996     

Misdivision analysis of centromere structure in maize.

The size and organization of a representative plant centromere from the supernumerary B chromosome were determined using a repeated sequence specific to the centric region. Several derivatives of the B chromosome that suffered from misdivision of the centromere were analyzed for the content and organization of their B repeat. In all these derivatives, major rearrangements were detected. Some misdivisions produced a significant reduction in size of the B-specific cluster. These results demonstrate that the B repeat is part of the functional centromere, that it is spread throughout its length, and that plant centromeres are composed of repeat units that can be significantly changed in copy number without a change in function.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Candida biotypes in patients with oral leukoplakia and lichen planus

Prevalence of yeasts in 35 leukoplakia and 34 oral lichen planus patients was compared with that observed in persons without oral diseases. Serotype and morphotype were determined on Candida albicans isolates. Yeasts were isolated from the oral cavity specimens of 43.7% of the patients. C. albicans (serotype A) was the predominant species (76% in leukoplakia, 88.2% in lichen planus and 60.8% in healthy persons). Sixteen morphotypes were encountered on malt extract agar, being 732, 733, 734, 753 and 754 the most frequently found. Morphotypes SP1N and SP1Y were the most common on Sabouraud-trypheniltetrazolium agar (68.4% of the isolates from leukoplakia and 73.3% from lichen planus, but only 46.6% of the isolates from healthy oral mucosa showed SP1N morphotype). Presence of oral lesions was associated with a marked reduction in the yeast species and C. albicans biotypes, suggesting that C. albicans and particularly some of its biotypes, show a high potential of adaptation to the changes associated with the development of oral leukoplakia and lichen planus.     

An extended x-ray absorption fine structure study of the high-affinity cation-binding site in the purple membrane.

The structure of the high-affinity cation-binding site of bacteriorhodopsin was studied using extended x-ray absorption fine structure techniques. The results obtained for Mn2+ in aqueous solution and for the complex BR-Mn2+ (1:1 molar ratio) show great similarities, suggesting that Mn2+, when bound to this site, is coordinated with six atoms of oxygen, forming an octahedral disposition. The interatomic distance between the atoms of oxygen and the Mn2+ was found to be 2.17 A for the complex BR-Mn2+, similar to Mn2+ in solution (2.15 A). In addition, the absence of any other peak at greater distances in the Fourier-transformed spectrum indicates that neither phosphorus nor sulphur atoms are present in the second coordination shell. This suggests that this binding site is located in the protein, discarding the proximity of lipid polar headgroups.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Narthecium ossifragum Associated Photosensitization in Sheep in The Faroe Islands

Hepatogenous photosensitization in sheep is an important problem in various parts of the world. Most photosensitization diseases are associated with ingestion of plant or fungal toxins. The lily, Narthecium ossifragum, has long been associated with photosensitization in lambs in western Norway (Ender 1955, Flåøyen 1993) and in the northern regions of the British Isles (Ford 1964).