Phosphoproteome analysis of Lotus japonicus roots reveals shared and distinct components of symbiosis and defense

Plant roots form an intracellular symbiosis with nitrogen-fixing bacteria while maintaining the capacity for defending themselves against bacterial pathogens. To investigate the molecular relationship between these opposing cellular responses, we compared changes in the root phosphoproteome of the legume Lotus japonicus occurring within minutes after perception of nodulation factor (NF), a symbiotic signaling molecule, to those elicited by flagellin peptide (flg22), a conserved pathogen-associated peptide motif present in flagellar protein of a wide range of bacteria. Phosphoproteins were visualized by autoradiography of two-dimensional polyacrylamide gels after in vivo labeling with 33P-orthophosphate. Comparisons of NF- and flg22-induced phosphoprotein patterns revealed signal-specific responses but also a surprisingly large overlap. Specificity of the responses was observed because the NF receptor kinases NFR1 and NFR5 were both required for NF- but not for flg22-mediated changes in the phosphoproteome. Moreover, NF did not stimulate an oxidative burst or activation of mitogen-activated protein kinases, two common markers for early defense responses that were induced by flg22. Inhibitor studies revealed that phosphorylation of at least some of the proteins in response to NF requires phospholipase D (PLD) whereas regulation of the flg22 phosphoproteome is PLD-independent. Although plant signal transduction during symbiosis and defense utilizes distinct components, phosphorylation of overlapping sets of proteins is achieved.     

A de novo protein binding pair by computational design and directed evolution

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.     

Simplified enrichment of plasma membrane proteins for proteomic analyses in Arabidopsis thaliana

The plasma membrane (PM) serves as the point of contact between cells and the outside environment. As such, changes in the PM proteome are an important component of understanding cellular responses to a diverse array of stimuli. However, intricate sample handling to enrich PM proteomes by traditional methods is both technically challenging and time consuming. Here, we describe a simplified method for decreasing the representation of other membrane-containing organelles such as the endoplasmic reticulum, plastids and mitochondria from crude microsomal membrane isolations. The decrease in other organellar proteomes results in an increase in both the total number of PM proteins and the number of spectra identified from these proteins representing the PM proteome. Therefore, this strategy represents a simple and rapid method for enriching PM proteins from Arabidopsis cell cultures for proteomic analyses.     

Organisms and responses to environmental change

There is great concern currently over environmental change and the biotic responses, actual or potential, to that change. There is also great concern over biodiversity and the observed losses to date. However, there has been little focus on the diversity of potential responses that organisms can make, and how this would influence both the focus of investigation and conservation efforts. Here emphasis is given to broad scale approaches, from gene to ecosystem and where a better understanding of diversity of potential response is needed. There is a need for the identification of rare, key or unique genomes and physiologies that should be made priorities for conservation because of their importance to global biodiversity. The new discipline of conservation physiology is one aspect of the many ways in which organismal responses to environmental variability and change can be investigated, but wider approaches are needed. Environmental change, whether natural or human induced occurs over a very wide range of scales, from nanometres to global and seconds to millennia. The processes involved in responses also function over a wide range of scales, from the molecular to the ecosystem. Organismal responses to change should be viewed in these wider frameworks. Within this overall framework the rate of change of an environmental variable dictates which biological process will be most important in the success or failure of the response. Taking this approach allows an equation to be formulated that allows the likely survival of future change to be estimated:Ps=(f(PF)xf(GM)xf(NP)xf(F)xf(D)xf(RA))/(ΔExf(C)xf(PR)xF(HS)),where Ps = Probability of survival; PF = Physiological flexibility; GM = Gene pool modification rate; NP = number in population; F = Fitness; D = Dispersal capability; RA = Resource availability; ΔE = rate of change of the environment; C = Competition; PR = Predation and parasitism; HS = Habitat separation. Functions (f) are used here to denote that factors may interact and respond in a non-linear fashion.     

Turning on the heat: ecological response to simulated warming in the sea

Significant warming has been observed in every ocean, yet our ability to predict the consequences of oceanic warming on marine biodiversity remains poor. Experiments have been severely limited because, until now, it has not been possible to manipulate seawater temperature in a consistent manner across a range of marine habitats. We constructed a "hot-plate" system to directly examine ecological responses to elevated seawater temperature in a subtidal marine system. The substratum available for colonisation and overlying seawater boundary layer were warmed for 36 days, which resulted in greater biomass of marine organisms and a doubling of space coverage by a dominant colonial ascidian. The "hot-plate" system will facilitate complex manipulations of temperature and multiple stressors in the field to provide valuable information on the response of individuals, populations and communities to environmental change in any aquatic habitat.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Adaptation and species range

Phase III of Sewall Wright's shifting-balance process involves the spread of a superior genotype throughout a structured population. However, a number of authors have suggested that this sort of adaptive change is unlikely under biologically plausible conditions. We studied relevant mathematical models, and the results suggest that the concerns about phase III of the shifting-balance process are justified, but only if environmental conditions are stable. If environmental conditions change in a way that alters species range, then phase III can be effective, leading to an enhancement of adaptedness throughout a structured population.     

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.     

IS LIFE IMPOSSIBLE? INFORMATION,SEX, AND THE ORIGIN OF COMPLEX ORGANISMS

The earliest organisms are thought to have had high mutation rates. It has been asserted that these high mutation rates would have severely limited the information content of early genomes. This has led to a well‐known “paradox” because, in contemporary organisms, the mechanisms that suppress mutations are quite complex and a substantial amount of information is required to construct these mechanisms. The paradox arises because it is not clear how efficient error‐suppressing mechanisms could have evolved, and thus allowed the evolution of complex organisms, at a time when mutation rates were too high to permit the maintenance of very substantial amounts of information within genomes. Here, we use concepts from the formal theory of information to calculate the amount of genomic information that can be maintained. We identify conditions under which much higher levels of genomic information can be maintained than previously considered possible among origin‐of‐life researchers. In particular, we find that the highest levels of information are maintained when many genotypes produce identical phenotypes, and when reproduction occasionally involves recombination between multiple parental genomes. There is a good reason to believe that these conditions are relevant for very early organisms, and thus the results presented may provide a solution to a long‐standing logical problem associated with the early evolution of life.