Diversity in and evidence for selection on the mitochondrial genome of Phytophthora infestans

Two extant nomenclature systems were reconciled to relate six mitochondrial DNA (mtDNA) haplotypes of Phytophthora infestans, the oomycete pathogen causing late blight disease on potato and tomato. Carter's haplotypes I-a and I-b were included in Goodwin's haplotype A, while Carter's haplotypes II-a and II-b were included in Goodwin's haplotype B. In addition, haplotypes E and F were included in Carter's haplotype I-b. The mutational differences separating the various haplotypes were determined, and we propose that either haplotype I-b(A) or haplotype I-a(A) is the putative ancestral mtDNA of P. infestans, because either can center all the other haplotypes in a logical stepwise network of mutational changes. The occurrence of the six haplotypes in 548 isolates worldwide was determined. Haplotypes I-a and II-a were associated with diverse genotypes worldwide. As previously suggested, haplotype I-b was found only in the US-1 clonal lineage and its variants (n = 99 isolates from 16 countries on 5 continents), and haplotype II-b was limited to the US-6 clonal lineage and its derivatives (n = 36). In a confirmation of a previous suggestion, the randomly mating population in the Toluca Valley of central Mexico (n = 78) was monomorphic for mtDNA haplotype I-a(A). We hypothesize that selection there may be driving the dominance of that single mtDNA haplotype.     

Interaction Energy Analysis of Nonclassical Antifolates with Human Dihydrofolate Reductase

The x-ray structure of the PTX:NADPH:L22F human mutant DHFR ternary complex was used as a structural template to generate structural models for the following wild type DHFR complexes: PTX:DHFR:NADPH, TMP:DHFR:NADPH, EPM:DHFR:NADPH, and TMQ:DHFR:NADPH. Each of these complexes were subsequently modeled in a 60 Å cube of explicit water and minimized to a rms gradient of from 1.0-3.0·10^-5 kcal·Å^-1. For each complex, interaction energies were calculated for the antifolate interaction with each of the following: the DHFR binding site residues, the entire DHFR protein, the solvated complex (containing DHFR, NADPH, and solvent water), water alone, and NADPH. Additionally, each antifolate was subdivided into distinct substructural regions and interaction energy calculations were performed in order to evaluate their contributions to overall antifolate interaction. Each antifolate showed its most stable interaction with the solvated complex. Substructural regions which consisted of a nitrogen containing aromatic ring system contributed most to the stability of the antifolate interactions, while the hydrocarbon aromatic rings, methoxy, and ethoxy groups showed much less stable interaction energies. Since the different substructural regions of nonclassical antifolates differ in their contributions to overall antifolate binding, those substructural regions which exhibit relatively unfavorable interaction energies may constitute important targets in the design of improved DHFR inhibitors.     

Effect of fire on the topsoil seed banks of rehabilitated bauxite mine sites in the jarrah forest of Western Australia

Summary Germinable seed stores of 5- and 8-year-old rehabilitated bauxite mine pits in south-west Western Australia were assessed before and after burning. These seed stores were compared to those of adjacent unmined Jarrah ( Eucalyptus marginata ) forest, to identify at what age fire can be reintroduced, in order to measure restoration success and reduce fire hazard. Soils were sampled in early summer (before fire) and late autumn (after fire). Before fire, the mean topsoil seed bank of 5-year-old sites was 2121 seeds per m² while 8-year-old sites had a mean of 1520 seeds per m². Only the 5-year-old sites were significantly different from the forest mean of 1478 seeds per m² for the same season. After summer burns (and possibly due to seasonal effects) topsoil seed banks of rehabilitated areas (sampled in autumn) decreased by an average of 53 per cent. Topsoil seed banks of 5–8-year-old sites were resistant to lower intensity burns, with 362 seeds per m² of native species surviving mild burns and 108 seeds per m² of native species surviving after an intense summer fire. The topsoil seed reserve of 5–8-year-old rehabilitated areas had a high proportion of annual weed species while the forest sites had high levels of subshrubs and native annuals. Low-intensity burns did not alter the composition of life-forms in the soil seed bank, while intense burns favoured annual weed and shrub species. The results indicate that it is not appropriate to introduce fire to rehabilitated areas before 8 years, due to limited fuel reduction benefits and possible adverse effects on obligate seeding species. The large proportion of weed species in the soil seed bank of young rehabilitated areas is a concern, and remains a major consideration for future disturbance of these areas.     

Simplified, Accurate Method for Antibiotic Assay of Clinical Specimens

Large glass plates are used for this modified agar-well diffusion assay method, allowing up to 81 replications on a single plate. With a specially designed agar punch, it is possible to prepare the small agar wells very quickly. The saving in serum resulting from fewer replications of standards with the large plates, and the small volume of the agar wells, makes it economically feasible to use pooled human serum for the standard antibiotic solutions. Methods are described for preparing the standard solutions, and for providing controls for the deterioration of standards and unknowns. Procedures for preparing and maintaining the commonly used assay organisms are presented. Serum specimens are tested directly rather than diluting them to a narrow range of antibiotic concentrations. This is possible because of a procedure for calculations that recognizes the curvilinear relationship between zone sizes and antibiotic concentrations. Adaptation of this method to a number of the commonly used antibiotics is described. With this method, it has been possible to test large numbers of clinical specimens in a minimal time, and with accuracy consistently better than 10%.     

Characterizing the socially transmitted foraging tactic “sponging” by bottlenose dolphins (Tursiops sp.) in the western gulf of Shark Bay,Western Australia

Individual foraging tactics are widespread in animals and have ecological and evolutionary implications. Indo‐Pacific bottlenose dolphins (Tursiops sp.) in Shark Bay, Western Australia, exhibit a foraging tactic involving tool use, called “sponging.” Sponging is vertically, socially transmitted through the matriline and, to date, has been described in detail in the eastern gulf of Shark Bay (ESB). Here, we characterize sponging in the western gulf of Shark Bay (WSB), in which a different matriline engages in the behavior. We identified 40 individual “spongers” in 9 mo of boat‐based surveys over three field seasons. As is the case in ESB, the majority of WSB spongers was female and engaged in sponging in deep channel habitats. In contrast to ESB, however, there was no difference in the number of associates between spongers and nonspongers in WSB, and activity budgets differed between spongers and deep‐water nonspongers; spongers foraged more frequently and rested less than nonspongers. Group sizes in deep channel habitat, where sponging was prevalent, were typically larger than those in shallow habitat, except for foraging, perhaps indicative of higher predator abundance and/or scattered prey distribution in deep‐water habitat. This research improves our understanding of within‐population foraging variations in bottlenose dolphins.     

Functional studies in living animals using multiphoton microscopy

In vivo microscopy is a powerful method for studying fundamental issues of physiology and pathophysiology. The recent development of multiphoton fluorescence microscopy has extended the reach of in vivo microscopy, supporting high-resolution imaging deep into the tissues and organs of living animals. As compared with other in vivo imaging techniques, multiphoton microscopy is uniquely capable of providing a window into cellular and subcellular processes in the context of the intact, functioning animal. In addition, the ability to collect multiple colors of fluorescence from the same sample makes in vivo microscopy uniquely capable of characterizing up to three parameters from the same volume, supporting powerful correlative analyses. Since its invention in 1990, multiphoton microscopy has been increasingly applied to numerous areas of medical investigation, providing invaluable insights into cell physiology and pathology. However, researchers have only begun to realize the true potential of this powerful technology as it has proliferated beyond the laboratories of a relatively few pioneers. In this article we present an overview of the advantages and limitations of multiphoton microscopy as applied to in vivo imaging. We also review specific examples of the application of in vivo multiphoton microscopy to studies of physiology and pathology in a variety of organs including the brain, skin, skeletal muscle, tumors, immune cells, and visceral organs.     

The N‐Terminal Sequences of Four Major Hydrogenosomal Proteins Are Not Essential for Import into Hydrogenosomes of Trichomonas vaginalis

The human pathogen Trichomonas vaginalis harbors hydrogenosomes, organelles of mitochondrial origin that generate ATP through hydrogen‐producing fermentations. They contain neither genome nor translation machinery, but approximately 500 proteins that are imported from the cytosol. In contrast to well‐studied organelles like Saccharomyces mitochondria, very little is known about how proteins are transported across the two membranes enclosing the hydrogenosomal matrix. Recent studies indicate that—in addition to N‐terminal transit peptides—internal targeting signals might be more common in hydrogenosomes than in mitochondria. To further characterize the extent to which N‐terminal and internal motifs mediate hydrogenosomal protein targeting, we transfected Trichomonas with 24 hemagglutinin (HA) tag fusion constructs, encompassing 13 different hydrogenosomal and cytosolic proteins of the parasite. Hydrogenosomal targeting of these proteins was analyzed by subcellular fractionation and independently by immunofluorescent localization. The investigated proteins include some of the most abundant hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO), which possesses an amino‐terminal targeting signal that is processed on import into hydrogenosomes, but is shown here not to be required for import into hydrogenosomes. Our results demonstrate that the deletion of N‐terminal signals of hydrogenosomal precursors generally has little, if any, influence upon import into hydrogenosomes. Although the necessary and sufficient signals for hydrogenosomal import recognition appear complex, targeting to the organelle is still highly specific, as demonstrated by the finding that six HA‐tagged glycolytic enzymes, highly expressed under the same promoter as other constructs studied here, localized exclusively to the cytosol and did not associate with hydrogenosomes.