Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.     

Keratella distribution in North Patagonian lakes (Argentina)

The distribution of Keratella species from 15 different lakes in North Patagonia (Argentina) was analysed. The genus was not present at altitudes above 1000 m. K. tropica was restricted to Patagonian Plateau lakes with a comparatively high conductivity. A morphometric analysis of the widely distributed K. cochlearis was performed. Results showed three groups of K. cochlearis corresponding to Andean lakes, Patagonian Plateau lakes and a Patagonian Reservoir.     

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.     

Effect of Sodium on Phosphate Uptake in Unicellular and Filamentous Cyanobacteria

The effect of Na⁺ on phosphate uptake was studied in four strainsof cyanobacteria: Synechococcus PCC 7942, Gloeothece PCC 6501,Phormidium sp. and Chlorogloeopsis PCC 6912. Phosphate uptakewas stimulated by Na⁺ in all cases. Li⁺ and K⁺ acted as partialanalogues for Na⁺. Half-saturation [K_1/2(Na⁺)] of phosphateuptake was reached with Na⁺ concentrations ranging from 317µM in Chlorogloeopsis to 659 µM in Phormidium. Theconcentration of phosphate required to reach half-saturationof phosphate uptake [K_1/2(P_i)]was not changed by the presenceof Na⁺. (Received April 11, 1994; Accepted July 5, 1994)     

Phagocytosis of Intravenously Administered Particles by Leukocytes Adhered to the Aortic Endothelium of the Rat

Phagocytosis has been used to characterize on a functional basis leukocytes adhered to the aortic endothelium of the rat. After intravenous administration of particles, phagocytosis was observed microscopically in esterase-positive leukocytes adhered to the endothelium in whole mounts of aorta. Polybead^R blue and red, 0.5 and 1 μm particle size, were inadequate because they were insufficiently colored to be identified individually at 400. Fluoresbrite^tm YG 0.25 and 0.50 μm at doses of 0.2 and 2 ± 0.3 m1/100 g, respectively, produced endothelial lesions. The same occurred with Monastral blue B^R (MbB) at 0.3 ml/100 g, red iron at 2 ± 16 mg/100 g and India ink at different concentrations depending on the supplier. At lower particle doses, lesions were not found. Deferoxamine mesylate 1.5 mg/100 g intravenous and allopurinol 5 mg/100 g intraperitoneal administered before the particles diminished the number and intensity of lesions. In none of the cases studied was the percentage of phagocytic cells greater than 50%. Clearance curves of MbB and Fluoresbrite^tm indicated rapid disappearance of particles from the blood. Results indicate that administration of particulate suspensions is not a good method for characterizing the phagocytic leukocytes adhering to the aortic endothelium because low doses produce rapid clearance of particles, thus impeding sufficient leukocyte loading, and higher doses produce endothelial lesions that often impair reliable counting of the adhering leukocytes.     

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.     

Acyl Group Migrations in 2-Monoolein

Acyl migration in 2-monoolein dissolved in solvents under conditions common in lipid modification reactions has been studied. The effects on acyl migration of solvent, incubation temperature, water activity, polar additives and solid additives have been investigated. Extensive acyl migration occured in aliphatic hydrocarbons and water-miscible alcohols under dry conditions. The acyl migration rate could be decreased in several nonpolar solvents by adding a small amount of water or an alcohol. Increasing water activity had no effect in isooctane, but decreased the acyl migration rate dramatically in methyl tert-butyl ether and methyl isobutyl ketone. Several commonly used enzyme supports catalysed acyl migration, showing that supports with surface charges could catalyse acyl migration.     

Effects of Auxin and Phenobarbital on Morphogenesis and Production of Digitoxin in Digitalis Callus

Pieces of callus obtained from seedlings of Digitalis purpureawere grown on solid Murashige-Skoog's medium supplemented with1 mg liter^–1 BA and 0.1 mg liter^–1 IAA or NAA, withor without phenobarbital (40 mg liter^–1). The replacementof the natural auxin IAA by the synthetic auxin NAA increasedcallus growth and inhibited organogenesis, whereas the additionof phenobarbital had the opposite effect. Morphometric measurementsrevealed a high ratio of vacuole to cytoplasm (v/v) in calluscells. This ratio was affected by the different treatments inthe same way as the fresh weight. The activity of mitochondrialcytochrome P450scc (the enzyme that provides the precursor,pregnenolone, for the biosynthesis of cardenolide in foxgloveplants) was detected in the relevant fraction of callus grownunder all experimental conditions, and its activity was increasedby the addition of phenobarbital. The different treatments testedincreased the cardenolide content and quantifiable amounts ofdigitoxin were detected in all callus tissues. It is of specialinterest that phenobarbital added to the culture medium increasedthe accumulation of digitoxin. The mechanism affecting the developmentand production of cardenolide in callus tissues of D. purpureaby phenobarbital and the replacement of IAA by NAA is discussed. (Received July 18, 1994; Accepted December 14, 1994)