Yeast UBL-UBA proteins have partially redundant functions in cell cycle control

Abp1, and the closely related Cbh1 and Cbh2 are homologous to the human centromere-binding protein CENP-B that has been implicated in the assembly of centromeric heterochromatin. Fission yeast cells lacking Abp1 show an increase in mini-chromosome instability suggesting that Abp1 is important for chromosome segregation and/or DNA synthesis. Here we show that Abp1 interacts with the DNA replication protein Cdc23 (MCM10) in a two-hybrid assay, and that the Δabp1 mutant displays a synthetic phenotype with a cdc23 temperature-sensitive mutant. Moreover, genetic interactions were also observed between abp1 ⁺ and four additional DNA replication initiation genes cdc18 ⁺, cdc21 ⁺, orc1 ⁺, and orc2 ⁺. Interestingly, we find that S phase is delayed in cells deleted for abp1 ⁺ when released from a G1 block. However, no delay is observed when cells are released from an early S phase arrest induced by hydroxyurea suggesting that Abp1 functions prior to, or coincident with, the initiation of DNA replication.     

The Solution Structure of Yeast tRNAPhe as Studied by Nuclear Overhauser Effects in NMR

Abstract

Recently, the imino proton spectrum of yeast tRNA^Phe has been assigned by means of the application of the nuclear Overhauser effect (NOE). In the present paper it will be shown that even for tRNA (MW 28000) connectivities between the imino proton spins can be observed using two-dimensional NOE spectroscopy. In this way the imino proton resonances of the D-stem region are assigned. The results are discussed in relation to those obtained by the classical one-dimensional nuclear Overhauser effect. It turns out that in 2D-NOE experiments connectivities from overlapping resonances can be observed which cannot be determined by one-dimensional Overhauser experiments. Moreover, the total assignment of the imino proton spectrum of yeast tRNA^Phe is used to relate the three-dimensional crystal structure of the tRNA to its solution structure. It is shown that the principle elements of the X-ray structure, i.e. the hydrogen bonding network and the stacking of the stems upon one another, are also found in solution. This is true for the presence as well as for the absence of magnesium ions. However, in absence of magnesium ions the tRNA structure appears to differ in details from that in the presence of magnesium ions. Finally, the influence of the elongation factor Tu from B.stearothermophilus on the tRNA structure is discussed.     

A “Prime-Pull” Vaccine Strategy Has a Modest Effect on Local and Systemic Antibody Responses to HIV gp140 in Mice

One potential strategy for the prevention of HIV infection is to induce virus specific mucosal antibody that can act as an immune barrier to prevent transmission. The mucosal application of chemokines after immunisation, termed “prime-pull”, has been shown to recruit T cells to mucosal sites. We wished to determine whether this strategy could be used to increase B cells and antibody in the vaginal mucosa following immunisation with an HIV antigen. BALB/c mice were immunised intranasally with trimeric gp140 prior to vaginal application of the chemokine CCL28 or the synthetic TLR4 ligand MPLA, without antigen six days later. There was no increase in vaginal IgA, IgG or B cells following the application of CCL28, however vaginal application of MPLA led to a significant boost in antigen specific vaginal IgA. Follow up studies to investigate the effect of the timing of the “pull” stimulation demonstrated that when given 14 days after the initial immunisation MPLA significantly increased systemic antibody responses. We speculate that this may be due to residual inflammation prior to re-immunisation. Overall we conclude that in contrast to the previously observed effect on T cells, the use of “prime-pull” has only a modest effect on B cells and antibody.     

Immunohistochemical,Ultrastructural and Functional Analysis of Axonal Regeneration through Peripheral Nerve Grafts Containing Schwann Cells Expressing BDNF,CNTF or NT3

We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB₄ or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.     

Induction of Malignant Plasma Cell Proliferation by Eosinophils

The biology of the malignant plasma cells (PCs) in multiple myeloma (MM) is highly influenced by the bone marrow (BM) microenvironment in which they reside. More specifically, BM stromal cells (SCs) are known to interact with MM cells to promote MM cell survival and proliferation. By contrast, it is unclear if innate immune cells within this same space also actively participate in the pathology of MM. Our study shows for the first time that eosinophils (Eos) can contribute to the biology of MM by enhancing the proliferation of some malignant PCs. We first demonstrate that PCs and Eos can be found in close proximity in the BM. In culture, Eos were found to augment MM cell proliferation that is predominantly mediated through a soluble factor(s). Fractionation of cell-free supernatants and neutralization studies demonstrated that this activity is independent of Eos-derived microparticles and a proliferation-inducing ligand (APRIL), respectively. Using a multicellular in vitro system designed to resemble the native MM niche, SCs and Eos were shown to have non-redundant roles in their support of MM cell growth. Whereas SCs induce MM cell proliferation predominantly through the secretion of IL-6, Eos stimulate growth of these malignant cells via an IL-6-independent mechanism. Taken together, our study demonstrates for the first time a role for Eos in the pathology of MM and suggests that therapeutic strategies targeting these cells may be beneficial.     

Global biogeography of highly diverse protistan communities in soil

Protists are ubiquitous members of soil microbial communities, but the structure of these communities, and the factors that influence their diversity, are poorly understood. We used barcoded pyrosequencing to survey comprehensively the diversity of soil protists from 40 sites across a broad geographic range that represent a variety of biome types, from tropical forests to deserts. In addition to taxa known to be dominant in soil, including Cercozoa and Ciliophora, we found high relative abundances of groups such as Apicomplexa and Dinophyceae that have not previously been recognized as being important components of soil microbial communities. Soil protistan communities were highly diverse, approaching the extreme diversity of their bacterial counterparts across the same sites. Like bacterial taxa, protistan taxa were not globally distributed, and the composition of these communities diverged considerably across large geographic distances. However, soil protistan and bacterial communities exhibit very different global-scale biogeographical patterns, with protistan communities strongly structured by climatic conditions that regulate annual soil moisture availability.     

The significance of overlapping plant range to a putative adaptive trade-off in the black bean aphid Aphis fabae Scop

Abstract. 1. This study continues to explore the analysis of a putative adaptive trade‐off in the utilisation of host plants Vicia faba and Tropaeolum majus by the aphid, Aphis fabae. These plants are utilised exclusively by the subspecies Aphis fabae fabae and A. f. mordwilkoi respectively, and this plant‐use system has been studied previously as a potential source of disruptive selection. 2. Here the potential of these two host plants to generate disruptive selection is considered given common utilisation of the abundant host plant, Rumex obtusifolius, by both subspecies. 3. The life history of subspecific clones is quantified in the laboratory on V. faba, T. majus, and R. obtusifolius at various temperatures and used to parameterise a temperature‐driven simulation model of aphid population development. 4. Accuracy of the model is tested using a field experiment, and fitness of clones on specific and common host is simulated using temperature data from a number of English sites. 5. The model gives a close quantitative fit to field data and makes the following predictions: performance of A. f. fabae is higher on the specific host than the common host under all tested thermal regimes; and performance of A. f. mordwilkoi is superior on the specific host in warm years but inferior in cold years. 6. Given the great abundance of R. obtusifolius relative to T. majus, the model predicts that the plant utilisation system has little potential to consistently promote hybrid dysfunction. This adds further weight to the assertion that the plant utilisation system studied can offer little insight into the evolutionary processes involved in subspecific differentiation and probably contains a host plant/host plants acquired after the evolution of reproductive barriers.     

Improving success of rare plant seed reintroductions: a case study of Dalea carthagenensis var. floridana,a rare legume with dormant seeds

Recent reviews of rare plant reintroduction success indicate that far fewer studies have been conducted with seeds than whole plants, and of these, less than 10% have established or had long‐term population persistence reported. Because seed reintroductions are relatively less expensive than plant reintroductions, determining ways to increase efficacy of using seeds to establish rare populations has conservation benefits. In laboratory trials, we examined seed germination of an endangered legume, Dalea carthagenensis var. floridana, endemic in South Florida, U.S.A. Laboratory treatments confirmed that seeds are hard seeded, remaining viable for 1,452 days even when moist; nicking, heat, and freezing triggered higher and more rapid germination than controls. Field trials begun in 2009, using pretreated (frozen) and untreated seeds within two habitats (natural and novel) revealed that freezing pretreatment increased germination in both habitats. However, plants matured, reproduced, and established seedlings only in natural habitat, not in novel habitat. By 2012, seed treatment plots in natural pine rockland had significantly greater numbers of reproductive plants and seedlings than controls. In a restoration context, using seed pretreatments to stimulate germination can improve establishment success in suitable habitats. When paired with essential vegetation management and a controlled burn, seed augmentation helped rescue the population from the brink of extinction.