A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.

A two-step centrifugation procedure has been developed to isolate greater quantities of highly purified sheep erythrocyte antigen-binding cells (ABC) than previously possible. The first step involves partially separating sheep erythrocyte rosettes from unrosetted lymphocytes by their difference in buoyant density on Ficoll-Hypaque. Subsequent passage through a linear 5 to 10% Ficoll gradient produces further purification of rosettes by sedimentation velocity. Approximately 4.5 X 10(6) ABC can be obtained at 50 to 100% purity from 10(9) immune spleen cells (5 days post-immunization) and 1 X 10(5) ABC at 20 to 40% purity from 10(9) nonimmune spleen cells. The purified ABC from 5-day immune animals are 80 to 90% B cells and 10 to 20% T cells, and represent between 30 and 40% of the original ABC in the spleen cell population. Less than 0.2% of the purified ABC are plaque-forming cells (PFC) and less than 2% have intracellular immunoglobulin (Ig) or J chain. The quantities of ABC obtained are sufficient for investigating biochemical parameters of antigen-induced lymphocyte activation and for direct analysis of the surface isotypes found on antigen-binding cells after immunization.     

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.     

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin alpha6beta1, but not the von Willebrand receptor GPIbalpha or the integrins alphaIIbbeta3 or alpha2beta1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.     

Is reproduction of endemic plant species particularly pollen limited in biodiversity hotspots?

Current evidence suggests that plants in biodiversity hotspots suffer more from pollen limitation of reproduction than those in lower diversity regions, primarily due to the response of self‐incompatible species. Species in biodiversity hotspots may thus be more at risk of limited reproduction and subsequent population decline. Should these species have restricted ranges (i.e. be endemics to a certain region), pollen limitation within highly diverse regions may pose an important threat to global plant biodiversity. We further dissect the global pattern by exploring whether pollen limitation of range‐restricted (endemic) species is distinctive and/or relates differently to species diversity than that of widespread (non‐endemic) species. To provide a preliminary test of this prediction we conducted both cross‐species and comparative phylogenetic meta‐analyses to determine the effect of endemism on the magnitude of pollen limitation and its relationship with regional species richness. Our data set included 287 plant species belonging to 78 families distributed world‐wide. Our results revealed that endemism and self‐compatibility contribute to the global association between pollen limitation and species richness. Self‐incompatible species were more pollen limited than self‐compatible ones, and the PICs analysis indicated that transitions to endemism were associated with transitions to self‐compatibility. The relationship between pollen limitation and species richness was significant only for the self‐incompatible species, and was monotonically increasing in non‐endemic species but accelerating in the endemic species. Thus, self‐incompatible endemic species from biodiversity hotspots are at the greatest risk of pollination failure, a previously unknown aspect suggesting this group of species as a top priority for future development of conservation strategies. In contrast, reproduction of self‐compatible species appears to be unrelated to plant diversity, although we caution that current data do not account for the reproductive limitation due to the quality of pollen received. Understanding the mechanisms underlying these patterns requires further investigation into plant–plant pollinator mediated interactions and the dynamics of pollen transfer in communities differing in species diversity.     

A quantitative synthesis of pollen supplementation experiments highlights the contribution of resource reallocation to estimates of pollen limitation

Our understanding of pollen limitation depends on a realistic view of its magnitude. Previous reviews of pollen supplementation experiments concluded that a majority of plant species suffers from pollen limitation and that its magnitude is high. Here, we perform a meta-analysis and find evidence that publication bias, experimental design, and the response variable chosen all influence the magnitude of pollen limitation. Fail-safe numbers indicate that publication bias exists for some measures of pollen limitation; significant results are more likely to be published and therefore available for review. Moreover, experiments conducted on only a fraction of a plant's flowers and reproductive episodes report ~8-fold higher effect sizes than those on all flowers produced over the entire lifetime, likely because resource reallocation among flowers and across years contributes to estimates of pollen limitation. Studies measuring percentage fruit set report higher values of pollen limitation than those measuring other response variables, such as seeds per fruit, perhaps because many plant species will not produce fruits unless adequate pollen receipt occurs to fertilize most ovules. We offer suggestions for reducing the bias introduced by methodology in pollen supplementation experiments and discuss our results in the context of optimality theory.     

Edaphic factors and plant–insect interactions: direct and indirect effects of serpentine soil on florivores and pollinators

Edaphic factors can lead to differences in plant morphology and tissue chemistry. However, whether these differences result in altered plant–insect interactions for soil-generalist plants is less understood. We present evidence that soil chemistry can alter plant–insect interactions both directly, through chemical composition of plant tissue, and indirectly, through plant morphology, for serpentine-tolerant Mimulus guttatus (Phrymaceae). First, we scored floral display (corolla width, number of open flowers per inflorescence, and inflorescence height), flower chemistry, pollinator visitation and florivory of M. guttatus growing on natural serpentine and non-serpentine soil over 2 years. Second, we conducted a common garden reciprocal soil transplant experiment to isolate the effect of serpentine soil on floral display traits and flower chemistry. And last, we observed arrays of field-collected inflorescences and potted plants to determine the effect of soil environment in the field on pollinator visitation and florivore damage, respectively. For both natural and experimental plants, serpentine soil caused reductions in floral display and directly altered flower tissue chemistry. Plants in natural serpentine populations received fewer pollinator visits and less damage by florivores relative to non-serpentine plants. In experimental arrays, soil environment did not influence pollinator visitation (though larger flowers were visited more frequently), but did alter florivore damage, with serpentine-grown plants receiving less damage. Our results demonstrate that the soil environment can directly and indirectly affect plant–mutualist and plant–antagonist interactions of serpentine-tolerant plants by altering flower chemistry and floral display.     

Sex ratio and subdioecy in Fragaria virginiana: the roles of plasticity and gene flow examined

Here we examined the roles of sex-differential plasticity (SDP) and gene flow in sex ratio evolution of subdioecious Fragaria virginiana. We assessed whether female frequency varied with resource availability in 17 natural populations and then characterized plasticity and mean investment in allocation to female function at flower and plant levels in the sex morphs in the glasshouse. We estimated patterns of population divergence using five microsatellite markers. We reveal SDP in fruit production substantial enough to translate into a higher equilibrium female frequency at low resources. Thus SDP can account, in part, for the strong negative relationship between female frequency and resources found in the field. Pollen-bearing morphs varied in plasticity across populations, and the degree of plasticity in fruit number was positively correlated with in situ variation in nitrogen (N) availability, suggesting an adaptive component to sex-allocation plasticity. Low neutral genetic differentiation, indicating high gene flow or recent divergence, may contribute to the absence of population differentiation in fruit-setting ability of pollen-bearing morphs despite considerable sex ratio variation. We consider how these processes, in addition to other features of this system, may work in concert to influence sex ratios and to hinder the evolution of dioecy in F. virginiana.