ALBERT

Notes: Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was 〉 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (〈 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated 〉 99.5% purity. The total non-P. carinii protein in the final preparation (〈 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 108-109 organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80–95%.

Notes: The use of teliospores of the rust fungus Pucdnia punctiformis (Str.) Röhl., a potential mycoher-bicide against the dicotyledonous weed Cirsium arvense (L.) Scop, has shown promise. Methods to increase teliospore production for systemic infections were investigated using two dicotyle-donous host-pathogen systems, the thistle rust and the bean rust (Phaseolus vulgaris-Uromyces appendiculatus). Three different approaches (culture filtrate extracts of different Aphano-fadium album strains, application of Ajoen and dark periods) were tested for their capacity to induce teliospore production in the above-mentioned host-pathogen systems.The methods significantly increased the teliospore production in the model system (bean-rust), although differing in the order dark period 〉 A. album extract ajoen. The effect of the A. album extract depends on the strain used.A two-day dark period increased the teliospore production of bean rust in addition to precipitating its onset. The same effect was noted for thistle rust using A. album extract. The advantage of bean rust as a model organism together with combined applications and host pathogen reaction mechanisms are discussed. La rouille du pois comme système modèle permettant d'évaluer l'efficacité de l'induction de téliosopores, en particulier chez le mycoherbicide potentiel Puccinia punctiformisL'utilisation de téliospores de la rouille Puccinia punctiformis (Str.) Röhl, un mycoherbicide potentiel, s'est montrée prometteuse pour la lutte contre la mauvaise herbe dicotylédone Cirsium arvense (L.) Scop. Des méthodes permettant d'augmenter la production de téliospores pour des infections systémiques ont étéétudiées sur deux systèmes hôte-pathogène de dicotylédones, la rouille du chardon et la rouille du haricot (Phaseolus vulgaris-Uromyces appendiculatus). Trois approches différentes (extraits de filtrats de culture provenant de différentes souches d'Aphanocladium album, application d'Ajoen et périodes obscures) ont été testées pour leur capacitéà induire les téliospores dans les systèmes hôte/pathogène précédents.Les méthodes accroissaient significativement la production de téliospores dans le système modèle (haricot-rouille) bien que différant en efficacité dans l'ordre: période obscure extraits d'A album Ajoen. L'effet de l'extrait d'A album dépendait de la souche utilisée.Une période obscure de deux jours accroissait la production de téliospores de la rouille du haricot et avanßait son démarrage. Le même effet était observé chez la rouille du chardon avec des extraits d'A album. L'avantage de la rouille du haricot comme organisme modèle, ainsi que d'applications combinées est discuté, de même que les mécanismes de la relation hôte/pathogène. Der Bohnenrost als Modellsystem zur Abschätzung der Effizienz unterschiedlicher Verfahren der Teleutosporeninduktion unter Berücksichtigung des potentiellen Mykoherbizids Puccinia punctiformisDie Verwendung der Teleutosporen des Rostes Puccinia punctiformis (Str.) Röhl. zeigt erfolgversprechende Ansätze als Mykoherbizid gegen das Unkraut Cirsium arvense (L.) Scop. Methoden, die für systemische Infektionen der Kratzdistel erforderliche Teleutosporenbildung zu steigern, wurden an 2 dikotylen Wirt-Pathosystemen, der Kratzdistel und der Bohne (Phaseolus vulgaris/Uromyces appendiculata), geprüft. Es wurden 3 unterschiedliche Methoden angewandt, um die Teleutosporenbildung in den erwähnten Systemen zu induzieren (Kulturfiltratextrakte unterschiedlicher Aphanocladium-album-Stämme, Ajoen und Dunkelperioden). Alle Anwendungen steigerten die Teleutosporenbildung im Modellsystem Bohnenrost signifikant, jedoch graduell unterschiedlich: Dunkelphase 〉 A.-album-Extrakt 〉 Ajoen. Dabei war der Effekt des A-album-Extraktes eindeutig von dem verwendeten Stamm abhängig. Die 2-tägige Dunkelphase steigerte die Teleutosporenproduktion des Bohnenrostes und bewirkte ein früheres Einsetzen der Teleutosporenbildung. Der gleiche Effekt konnte für den Kratzdistelrost auch unter Verwendung des A.-album-Extraktes festgestellt werden. Die Vorteile des Bohnenrostes als Modellorganismus sowie kombinierte Appli-kationen und mögliche Wirt-Pathogen-Reaktions-mechanismen werden diskutiert.

Notes: Abstract Bacillus cells frequently faced with various adverse environmental factors in nature have evolved different adaptational strategies. The induction of stress proteins is an essential component of this adaptational network. In Bacillus subtilis there are two groups of stress proteins. The first group is factor specific, whereas the second group is induced by growth restrictive conditions in general. The relationship between the stringent response and the induction of stress proteins is discussed.

Notes: Abstract Heat-inducible DNA fragments of Bacillus subtilis were cloned with two different promoter probe vectors. The increased synthesis of the reporter enzymes seemed to be due to a transient increase in the transcription of the encoding genes. The structures of the heat-sensitive promoters resembles the consensus sequence of promoters recognized by the vegetative form of RNA polymerase of B. subtilis. Our results support data in literature that the heat shock response of B. subtilis is regulated by a different mechanism than in Escherichia coli, where alternative sigma factors direct the transcription of heat shock genes.

Notes: Summary A major deficit in our understanding of membrane biogenesis in eukaryotes is the definition of mechanisms by which the lipid constituents of cell membranes are transported from their sites of intracellular synthesis to the multiplicity of membranes that constitute a typical cell. A variety of approaches have been used to examine the transport of lipids to different organelles. In many cases the development of new methods has been necessary to study the problem. These methods include cytological examination of cells labeled with fluorescent lipid analogs, improved methods of subcellular fractionation, in situ enzymology that demonstrates lipid translocation by changes in lipid structure, and cell-free reconstitution with isolated organelles. Several general patterns of lipid transport have emerged but there does not appear to be a unifying mechanism by which lipids move among different organelles. Significant evidence now exists for vesicular and metabolic energy-dependent mechanisms as well as mechanisms that are clearly independent of cellular ATP content.

Notes: Abstract Normal rat kidney cells, non-productively infected with the anaemia-inducing variant of Friend spleen focus-forming virus (F-SFFVA), were metabolically labelled with [2-3H]mannose. The primary translation product of the viral envelope gene (env), representing a glycoprotein with an apparent molecularM r of 55 000 (gp55), was isolated from cell lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Radiolabelled oligosaccharides, released from tryptic glycopeptides by treatment with endo-β-N-acetylglucosaminidase H, were characterized chromatographically, by enzymic digestion and by acetolysis. The results revealed that F-SFFVA gp55 obtained from this source carried predominantly oligomannose type sugar chains with five to nine mannoses. As a characteristic feature, glycans with seven to nine mannoses contained, in part, an additional glucose residue. Although the amount of glucosylated species found was higher in F-SFFVA gp55 (about 25% of total endo-H-sensitive oligosaccharides) than in gp55 of the corresponding polycythaemia-inducing variant (F-SFFVP, 16.3%), the overall glycosylation pattern of the F-SFFVA env product closely resembled that of F-SFFVP gp55 [Strubeet al. (1988)J Biol Chem 263:3762–71]. Hence, our results demonstrate that the different intracellular processing and transport of the primary F-SFFVA env product cannot be attributed to aberrant trimming of its oligomannose type glycans.

Notes: Summary The nucleotide sequence of the Escherichia coli K12 β-methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome.