A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Relative Values of Physiological Parameters of P Response of Different Genotypes can be Measured in Experiments with Only Two P Treatments

Improved methods for selecting cultivars for their ability to grow well on low P soils will improve productivity and minimise pollution. Estimates are required of the relative values for different genotypes of physiologically meaningful parameters that define yield response to increasing levels of plant available soil P. The object of this study was to devise ways of obtaining these estimates from conventional trials that include numerous genotypes but only two P treatments. The parameters were estimated as coefficients of a modified Michaelis–Menten equation, namely A, the maximum yield that can be obtained with ample P, Km, the concentration of plant available P in the rooting medium at which yield is half the maximum, and B, the gradient of yield against available P as it approaches the origin. Two novel methods of estimation were devised. They both require inputs of yield and its variance at each of two concentrations of available P, but these must be within certain limits. Estimates of parameter values for 12 Brassica oleracea genotypes made by these methods from two P-levels in each of two experiments with six P levels were compared with the values obtained by conventional fitting to the data from all six P levels. According to regression and ranking analyses, the relative values of A obtained for the different genotypes by both two P-level methods were similar to those from the six P-level method and both were reproducible. None of the methods however detected any reproducible differences in Km. Application of one of the two P-level methods to published data for genotypes of other species indicated that there was little intra-species variation in Km within some data sets on maize, beans and leguminous cover crops but much within other data sets. Sensitivity and algebraic analyses are presented to define the experimental conditions required for successful use of two P-level methods. It is concluded that, provided these experimental conditions are met, measurements at two P levels can give almost as much information about intra-species differences in P response parameters as measurements at six P levels. The procedure may also be useful for interpreting other responses of a “diminishing returns” type such as those to other nutrients or plant spacing.     

STOMACH CONTENTS OF LONG-FINNED PILOT WHALES (GLOBICEPHALA MELAS) STRANDED ON THE U.S. MID-ATLANTIC COAST

Ten prey taxa were recorded from the stomach contents of eight long-finned pilot whales ( Globicepbala melas ) independently stranded along the U.S. mid-Atlantic coast. Relative importance of prey species was determined by methods that incorporate prey frequencies of occurrence, proportions of numerical abundance, and proportions of reconstructed mass. Separate analyses of trace (free, durable body parts representing well-digested prey items) and non-trace (relatively intact prey specimens) food material were conducted in order to address biases caused by differential rates of digestion and passage through the gastrointestinal tract. Different measures of prey importance yielded varying results, but the long-finned squid ( Loligo pealei ) was the most important prey species regardless of how prey importance was defined. Fishes were relatively unimportant in the diet. Our results indicate that the diets of western North Atlantic long-finned pilot whales differ substantially from what has been previously reported in the literature and that results from food-habits studies that utilize different techniques may not be comparable.     

Differentiation of Staphylococcus aureus from freshly slaughtered poultry and strains 'endemic'to processing plants by biochemical and physiological tests

A comparison was made of 27 'endemic' strains of Staphylococcus aureus and 35 strains from freshly slaughtered birds, isolated at five commercial slaughterhouses processing chickens or turkeys. Of 112 biochemical and physiological tests used, 74 gave results which differed among the strains. Cluster analysis revealed several distinct groupings which were influenced by strain type, processing plant and bird origin; these included a single group at the 72% level of similarity containing most of the 'endemic' strains. In comparison with strains from freshly slaughtered birds, a higher proportion of 'endemic' strains produced fibrinolysin, aP-glucosidase and urease and were β-haemolytic on sheep-blood agar. The 'endemic' type also showed a greater tendency to coagulate human but not bovine plasma, and to produce mucoid growth and clumping. The last two properties, relevant to colonization of processing equipment, were less evident in heart infusion broth than in richer media or process water collected during defeathering of the birds.     

The metabolism of pyrimidines by proteolytic clostridia

Uracil was used by growing cultures of Clostridium sporogenes, and by proteolytic strains of C. botulinum types A and B. Uracil was not used by C. bifermentans; C. botulinum, type B (non-proteolytic); C. botulinum, type F (non-proteolytic); C. botulinum, type E; C. butyricum; C. cochlearium; C. difficile; C. histolyticum; C. oedematiens, type A; C. paraputrificum; C. scatologenes; C. septicum; C. sordellii; C. sticklandii; C. tertium; C. tetani; C. tetanomorphum; C. welchii, types A, B, C, E and 4 untyped strains. The growth of C. sporogenes was not increased by uracil; it was reduced to dihydrouracil. Experiments with washed cells of C. sporogenes showed that the uracil-reducing system was inducible. Washed cell suspensions incubated under hydrogen with uracil, thymine, iso-barbituric acid, 5-amino uracil and cytosine consumed 1 mole H₂/mole pyrimidine. The reduction product of cytosine was dihydrouracil indicating that it was deaminated before reduction. The reduction products of the remaining pyrimidines were the corresponding dihydro derivatives. Extracts of C. sporogenes reduced uracil in the presence of NADPH₂ but not NADH₂.     

Biosynthesis of long chain fatty acids by oligodendroglia isolated from bovine white matter.

Abstract— Radioactively labelled fatty acids were incubated with interfascicular oligodendroglial preparations isolated from 9 month fetal and adult bovine CNS white matter to study their metabolism by these cells. Of the various acids studied, the uptake was greatest for palmitic acid and decreased with decreasing chain length. Laurie acid was converted to the greatest extent to other fatty acids. The incorporation of oleic and linoleic acids in the oligodendroglia from both the fetal and adult brains was higher than that of linolenic acid. Fatty acids underwent chain elongation, desaturation and oxidation. Oleic acid was elongated to nervonic acid. Fatty acids were incorporated into both cerebrosides and phospholipids, with preferential incorporation into ethanolamine phosphoglyceride.