Origins of host-specific populations of the blast pathogen Magnaporthe oryzae in crop domestication with subsequent expansion of pandemic clones on rice and weeds of rice

Rice, as a widely and intensively cultivated crop, should be a target for parasite host shifts and a source for shifts to co-occurring weeds. Magnaporthe oryzae, of the M. grisea species complex, is the most important fungal pathogen of rice, with a high degree of host specificity. On the basis of 10 loci from six of its seven linkage groups, 37 multilocus haplotypes among 497 isolates of M. oryzae from rice and other grasses were identified. Phylogenetic relationships among isolates from rice (Oryza sativa), millet (Setaria spp.), cutgrass (Leersia hexandra), and torpedo grass (Panicum repens) were predominantly tree like, consistent with a lack of recombination, but from other hosts were reticulate, consistent with recombination. The single origin of rice-infecting M. oryzae followed a host shift from a Setaria millet and was closely followed by additional shifts to weeds of rice, cutgrass, and torpedo grass. Two independent estimators of divergence time indicate that these host shifts predate the Green Revolution and could be associated with rice domestication. The rice-infecting lineage is characterized by high copy number of the transposable element MGR586 (Pot3) and, except in two haplotypes, by a loss of AVR-Co39. Both mating types have been retained in ancestral, well-distributed rice-infecting haplotypes 10 (mainly temperate) and 14 (mainly tropical), but only one mating type was recovered from several derived, geographically restricted haplotypes. There is evidence of a common origin of both ACE1 virulence genotypes in haplotype 14. Host-haplotype association is evidenced by low pathogenicity on hosts associated with other haplotypes.     

Genetic variation for total fitness in Drosophila melanogaster: complex yet replicable patterns

The extent of genetic variation in fitness is a crucial issue in evolutionary biology and yet remains largely unresolved. In Drosophila melanogaster, we have devised a method that allows the net effects on fitness of heterozygous wild-type chromosomes to be measured, by competing them against two different "balancer" chromosomes. We have applied the method to a large sample of 40 wild-type third chromosomes and have measured fitnesses of nonlethal chromosomes as well as chromosomes bearing recessive lethals. The measurements were made in the environment to which the population was adapted and did not involve inbreeding. The results show an extraordinary similarity in the behavior of replicates of the same chromosome, indicating consistent genetic effects on total fitness. Some invading chromosomes increased rapidly and some slowly, and some rose to appreciable frequency after several months, but then declined again: in every case, the same pattern was seen in each replicate. We estimated relative fitnesses, rates of change of fitness, and relative viabilities, for each chromosome. There were significant fluctuations around the fitted model, which were also highly replicable. Wild-type chromosomes varied substantially in their effects on heterozygous fitness, and these effects vary through time, most likely as a result of genotype x environment interactions.     

Photophysics of tryptophan fluorescence: link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum

The formation of all major intermediates in the reaction catalyzed by the citrate synthase from Thermoplasma acidophilum is accompanied by changes in tryptophan fluorescence. The largest change is the strong quenching observed on formation of the binary complex with substrate, oxaloacetate (OAA). The four tryptophan residues present in the enzyme have been changed to nonfluorescent ones in various combinations without major perturbations in protein stability, enzyme mechanism, or other physical properties. W348, residing in the hydrophobic core of the protein behind the active site wall ca. 9 A from OAA, is responsible for the majority of the protein's intrinsic fluorescence and all of the quenching that accompanies OAA binding. Lifetime studies show that all of the quenching results from excited-state processes. The lack of solvent isotope effects on the quantum yields excludes a quenching mechanism involving proton transfer to an acceptor. There are no significant changes in fluorescence properties in single site mutants of residues near W348 that change conformation and/or interactions when OAA binds. This result excludes these changes from a direct role. Electron transfer from the indole excited state to some acceptor is the major quenching mechanism; the reduced quenching observed in the 5F-W-substituted protein strengthens this conclusion. Using the X-ray structures of the unliganded enzyme and its OAA binary complex, hybrid quantum mechanics-molecular dynamics (QM-MM) calculations show that OAA itself is the most likely quencher with the OAA carbonyl as the electron acceptor. This conclusion is strengthened by the ability of an alpha-keto acid model compound, trimethylpyruvate, to act as a diffusional quencher of indole fluorescence in solution. The theoretical calculations further indicate that the positive electrostatic potential surrounding the OAA carbonyl within the enzymes' active site is essential to its ability to accept an electron from the excited state of W348. These same environmental factors play a major role in activating OAA to react with the carbanion of acetyl-CoA. Since carbonyl polarization plays a role in the catalytic strategies of numerous enzymes whose reactions involve this functional group, tryptophan fluorescence changes might be useful as a mechanistic probe for other systems.     

Main chain and side chain dynamics of the ubiquitin conjugating enzyme variant human Mms2 in the free and ubiquitin-bound States

Protein ubiquitination involves a cascade of enzymatic steps where ubiquitin (Ub) is sequentially transferred as a thiolester intermediate from an E1 enzyme to an E2 enzyme and finally to the protein target with the help of a Ub-protein ligase. Protein ubiquitination brought about by the Ubc13-Mms2 (E2-E2) complex has a unique role in the cell, unrelated to protein degradation. The Mms2-Ubc13 heterodimer links Ub molecules to one another through an isopeptide bond between its own C-terminus and Lys-63 on another Ub. The role of Mms2 is to orient a target-bound Ub molecule such that its Lys-63 is proximal to the C-terminus of the Ub molecule that is covalently linked to the active site of Ubc13. To gain insight into the influence of protein dynamics on the affinity of Ub for Mms2, we have determined pico- to nanosecond time scale fluctuations of the main chain and methyl side chains of human Mms2 in the free and Ub-bound states using solution state (15)N and (2)H nuclear magnetic resonance relaxation measurements. Analysis of the relaxation data allows for a semiquantitative estimation of the conformational entropy change (TDeltaS(NMR)) for the main chain and side chain methyl groups of Mms2 upon binding Ub. The value of TDeltaS(NMR) for the main chain and side chain methyl groups of Mms2 is -8 +/- 2 and -2 +/- 2 kcal mol(-)(1), respectively. The experimental DeltaG(binding) for the Mms2.Ub complex is -6 kcal mol(-)(1). Estimation of DeltaG(binding) using an empirical structure-based approach that does not account for changes in main chain entropy yields a value of -17 +/- 2 kcal mol(-)(1). However, inclusion of TDeltaS(NMR) for the main chain of Mms2 increases the estimated DeltaG(binding) to -9 +/- 3 kcal mol(-)(1). Assuming that changes in Ub main chain dynamics contribute to TDeltaS(NMR) to the same extent as Mms2, the estimated DeltaG(binding) is further reduced to -1 +/- 4 kcal mol(-)(1), a value close to the experimental DeltaG(binding).     

Philosophy of Biology: Outline of a Transcendental Project

This paper analyses the actual meaning of a transcendental philosophy of biology, and does so by exploring and actualising the epistemological and metaphysical value of Kant's viewpoint on living systems. It finds inspiration in the Kantian idea of living systems intrinsically resisting objectification, but critically departs from Kant's philosophical solution in as far as it is based in a subjectivist dogmatism. It attempts to overcome this dogmatism, on the one hand by explicitly taking into account the conditions of possibility at the side of the subject, and on the other hand by embedding both the living and the knowing system into an ontology of complexly organized dynamical systems. This paper fits into the transcendental perspective in acknowledging the need to analyse the conditions of knowability, prior to the contents of what is known. But it also contributes to an expansion and an actualisation of the issue of transcendentality itself by considering the conditions of possibility at the side of the object as intrinsically linked to the conditions of possibility at the side of the subject.     

Only seed size matters for germination in different populations of the dimorphic Tragopogon pratensis subsp. pratensis (Asteraceae)

Many studies have focused on the ecology of seed dimorphism, the production of two seed types by a single plant. Morphology and seed size are usually correlated, but how morphology affects germination percentage and seedling growth is poorly understood. Here we explicitly separate these effects for nine populations of the dimorphic species Tragopogon pratensis subsp. pratensis. Larger seeds yielded higher germination percentages, yet seed morphology had no additional direct effect on germination. Neither seed size nor seed morphology affected seedling growth. Neither germination nor seedling growth varied among populations, but seed head varied significantly. Results show that germination is mainly controlled by seed size rather than by seed morphology. This study is one of the few to distinguish explicitly between seed size and seed morphology effects on ecological characteristics and suggests that seed dimorphism may exert its ecological effects predominantly through its correlated size.     

Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.     

The African swine fever virus dynein-binding protein p54 induces infected cell apoptosis

A specific interaction of ASFV p54 protein with 8 kDa light chain cytoplasmic dynein (DLC8) has been previously characterized and this interaction is critical during virus internalization and transport to factory sites. During early phases of infection, the virus induces the initiation of apoptosis triggering activation of caspase-9 and -3. To analyze the role of the structural protein p54 in apoptosis, transient expression experiments of p54 in Vero cells were carried out which resulted in effector caspase-3 activation and apoptosis. Interestingly, p54 mutants, lacking the 13 aa dynein-binding motif lose caspase activation ability and pro-death function of p54. This is the first reported ASFV protein which induces apoptosis.