The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.     

CPMG sequences with enhanced sensitivity to chemical exchange

Improved relaxation-compensated Carr–Purcell–Meiboom-Gill pulse sequences are reported for studying chemical exchange of backbone ¹⁵N nuclei. In contrast to the original methods [J. P. Loria, M. Rance, and A. G. Palmer, J. Am. Chem. Soc. 121, 2331–2332 (1999)], phenomenological relaxation rate constants obtained using the new sequences do not contain contributions from ¹H-¹H dipole-dipole interactions. Consequently, detection and quantification of chemical exchange processes are facilitated because the relaxation rate constant in the limit of fast pulsing can be obtained independently from conventional ¹⁵N spin relaxation measurements. The advantages of the experiments are demonstrated using basic pancreatic trypsin inhibitor.     

Strong induction of tyrosine phosphorylation,intracellular calcium,nuclear transcription factors and interferongamma,but weak induction of IL-2 in naïve T cells stimulated by bacterial superantigen

The outcome of T cell receptor (TCR) engagement is controlled by the differential recruitment of a variety of pathways, depending on the nature of the TCR ligand. Studies on superantigens (SAGs) were among the first describing such differential signaling; however, reported results are inconsistent. We took a quantitative approach to reinvestigate this question. Using nai;ve T cells from TCR transgenic mice, we found that compared to the antigenic peptide from pigeon cytochrome c, the SAG staphylococcal enterotoxin A very efficiently (100-2000-fold more sensitive on a weight basis) induced tyrosine kinase activity, intracellular calcium increase, and interferon (IFN)gamma production. Up-regulation of CD25 and CD69 and proliferation were less efficiently induced (20-30-fold more sensitive), and interleukin (IL)-2 production was induced least efficiently (only 2-fold more sensitive). This differential activation profile that varies with the activation event analyzed is discussed with respect to the propensity for SAG to induce anergy.     

Benefits and costs of mutualism: demographic consequences in a pollinating seed-consumer interaction

Interspecific interactions can affect population dynamics and the evolution of species traits by altering demographic rates such as reproduction and survival. The influence of mutualism on population processes is thought to depend on both the benefits and costs of the interaction. However, few studies have explicitly quantified both benefits and costs in terms of demographic rates; furthermore there has been little consideration as to how benefits and costs depend on the demographic effects of factors extrinsic to the interaction. I studied how benefits (pollination) and costs (larval fruit consumption) of pollinating seed-consumers (senita moths) affect the reproduction of senita cacti and how these effects may rely on extrinsic water limitation for reproduction. Fruit initiation was not limited by moth pollination, but survival of initiated fruit increased when moth eggs were removed from flowers. Watered cacti produced more flowers and initiated more fruit from hand-pollinated flowers than did unwatered cacti, but fruit initiation remained low despite excess pollen. Even though water, pollination and larvae each affected a component of cactus reproduction, when all of these factors were included in a factorial experiment, pollination and water determined rates of reproduction. Counter-intuitively, larval fruit consumption had a negligible effect on cactus reproduction. By quantifying both benefits and costs of mutualism in terms of demographic rates, this study demonstrates that benefits and costs can be differentially influential to population processes and that interpretation of their influences can depend on demographic effects of factors extrinsic to the interaction.     

Ecological and evolutionary conditions for fruit abortion to regulate pollinating seed-eaters and increase plant reproduction

Coevolved mutualisms, such as those between senita cacti, yuccas, and their respective obligate pollinators, benefit both species involved in the interaction. However, in these pollination mutualisms the pollinator's larvae impose a cost on plants through consumption of developing seeds and fruit. The effects of pollinators on benefits and costs are expected to vary with the abundance of pollinators, because large population sizes result in more eggs and larval seed-eaters. Here, we develop the hypothesis that fruit abortion, which is common in yucca, senita, and plants in general, could in some cases have the function of limiting pollinator abundance and, thereby, increasing fruit production. Using a general steady-state model of fruit production and pollinator dynamics, we demonstrate that plants involved in pollinating seed-eater mutualisms can increase their fecundity by randomly aborting fruit. We show that the ecological conditions under which fruit abortion can improve plants fecundity are not unusual. They are best met when the plant is long-lived, the population dynamics of the pollinator are much faster than those of the plant, the loss of one fruit via abortion kills a larva that would have the expectation of destroying more than one fruit through its future egg laying as an adult moth, and the effects of fruit abortion on pollinator abundance are spatially localized. We then use the approach of adaptive dynamics to find conditions under which a fruit abortion strategy based on regulating the pollinator population could feasibly evolve in this type of plant-pollinator interaction.     

Normal mode analysis of macromolecular motions in a database framework: developing mode concentration as a useful classifying statistic

We investigated protein motions using normal modes within a database framework, determining on a large sample the degree to which normal modes anticipate the direction of the observed motion and were useful for motions classification. As a starting point for our analysis, we identified a large number of examples of protein flexibility from a comprehensive set of structural alignments of the proteins in the PDB. Each example consisted of a pair of proteins that were considerably different in structure given their sequence similarity. On each pair, we performed geometric comparisons and adiabatic-mapping interpolations in a high-throughput pipeline, arriving at a final list of 3,814 putative motions and standardized statistics for each. We then computed the normal modes of each motion in this list, determining the linear combination of modes that best approximated the direction of the observed motion. We integrated our new motions and normal mode calculations in the Macromolecular Motions Database, through a new ranking interface at http://molmovdb.org. Based on the normal mode calculations and the interpolations, we identified a new statistic, mode concentration, related to the mathematical concept of information content, which describes the degree to which the direction of the observed motion can be summarized by a few modes. Using this statistic, we were able to determine the fraction of the 3,814 motions where one could anticipate the direction of the actual motion from only a few modes. We also investigated mode concentration in comparison to related statistics on combinations of normal modes and correlated it with quantities characterizing protein flexibility (e.g., maximum backbone displacement or number of mobile atoms). Finally, we evaluated the ability of mode concentration to automatically classify motions into a variety of simple categories (e.g., whether or not they are "fragment-like"), in comparison to motion statistics. This involved the application of decision trees and feature selection (particular machine-learning techniques) to training and testing sets derived from merging the "list" of motions with manually classified ones.     

Survival analysis using auxiliary variables via multiple imputation, with application to AIDS clinical trial data

We develop an approach, based on multiple imputation, to using auxiliary variables to recover information from censored observations in survival analysis. We apply the approach to data from an AIDS clinical trial comparing ZDV and placebo, in which CD4 count is the time-dependent auxiliary variable. To facilitate imputation, a joint model is developed for the data, which includes a hierarchical change-point model for CD4 counts and a time-dependent proportional hazards model for the time to AIDS. Markov chain Monte Carlo methods are used to multiply impute event times for censored cases. The augmented data are then analyzed and the results combined using standard multiple-imputation techniques. A comparison of our multiple-imputation approach to simply analyzing the observed data indicates that multiple imputation leads to a small change in the estimated effect of ZDV and smaller estimated standard errors. A sensitivity analysis suggests that the qualitative findings are reproducible under a variety of imputation models. A simulation study indicates that improved efficiency over standard analyses and partial corrections for dependent censoring can result. An issue that arises with our approach, however, is whether the analysis of primary interest and the imputation model are compatible.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

The antithyroid agent 6-n-propyl-2-thiouracil is a mechanism-based inactivator of the neuronal nitric oxide synthase isoform

6-n-Propyl-2-thiouracil (6-PTU), the antithyroid agent, produces a time-, concentration-, and turnover-dependent inactivation of the NO synthetic capability of the neuronal nitric oxide synthase isoform irreversible by either arginine or (6R)-5,6,7,8-tetrahydro-L-biopterin. By contrast 6-PTU produces an inhibition of the cytokine-inducible and endothelial nitric oxide synthases fully reversible by arginine. The inactivation of neuronal nitric oxide synthase by 6-PTU follows first order kinetics, and is inhibited competitively by both arginine and (6R)-5,6,7,8-tetrahydro-L-biopterin, but is not accompanied by either a loss of heme-CO binding, heme fluorescence, or disassembly of dimeric structure. 2-Thiouracil behaves qualitatively identically to 6-PTU. Turnover-dependent inactivation of neuronal nitric oxide synthase by [2-14C]-2-thiouracil is accompanied by incorporation of radioactivity into the polypeptide chain. Ca2+-dependent NO formation by GH3 pituitary cells is inhibited by 6-PTU in a manner enhanced by depletion of either extracellular arginine or intracellular (6R)-5,6,7,8-tetrahydro-L-biopterin. These observations establish that 6-PTU is an alternate substrate, mechanism-based inactivator of the neuronal nitric oxide synthase isoform with the ability to suppress cellular NO formation.     

Historical contingency in the evolution of primate color vision

Primates are unique among eutherian mammals for possessing three types of retinal cone. Curiously, catarrhines, platyrrhines, and strepsirhines share this anatomy to different extents, and no hypothesis has hitherto accounted for this variability. Here we propose that the historical biogeography of figs and arborescent palms accounts for the global variation in primate color vision. Specifically, we suggest that primates invaded Paleogene forests characterized by figs and palms, the fruits of which played a keystone function. Primates not only relied on such resources, but also provided high-quality seed dispersal. In turn, figs and palms lost or simply did not evolve conspicuous coloration, as this conferred little advantage for attracting mammals. We suggest that the abundance and coloration of figs and palms offered a selective advantage to foraging groups with mixed capabilities for chromatic distinction. Climatic cooling at the end of the Eocene and into the Neogene resulted in widespread regional extinction or decimation of palms and (probably) figs. In regions where figs and palms became scarce, we suggest primates evolved routine trichromatic vision in order to exploit proteinaceous young leaves as a replacement resource. A survey of the hue and biogeography of extant figs and palms provides some empirical support. Where these resources are infrequent, primates are routinely trichromatic and consume young leaves during seasonal periods of fruit dearth. These results imply a link between the differential evolution of primate color vision and climatic changes during the Eocene-Oligocene transition.