Potential effects of plant protease inhibitors, oryzacystatin I and soybean Bowman-Birk inhibitor, on the aphid parasitoid Aphidius ervi Haliday (Hymenoptera, Braconidae)

Protease inhibitors (PIs) have been shown to cause lethal and sublethal effects on aphids depending on the kind of PI and aphid species. Therefore, these proteins might affect aphid parasitoids directly by inhibiting their digestive proteolysis or indirectly via their development in a less suitable host. In our study, the risk of exposure and the potential effects of soybean Bowman-Birk inhibitor (SbBBI) and oryzacystatin I (OCI) on the aphid endoparasitoid Aphidius ervi were investigated using artificial diet to deliver PIs. Immunoassays showed that both SbBBI and OCI were detected in the honeydew of aphids reared on artificial diet containing these recombinant proteins at 100 microg/mL. However, only SbBBI was detected in parasitoid larvae, while this PI could not be detected in adult parasitoids emerged from PI-intoxicated aphids. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of A. ervi predominantly relies on serine proteases and especially on chymotrypsin-like activity. Bioassays using SbBBI and OCI on artificial diet were performed. A. ervi that developed on intoxicated aphids had impaired fitness. Thus development and parasitism success of parasitoids exposed to OCI were severely affected. On the contrary, SbBBI only altered significantly female size and sex ratio. Direct exposure to PIs through adult food intake did not affect female's longevity, while SbBBI and OCI (100 microg/mL) induced 69% and 30% inhibition of digestive protease activity, respectively. These studies made it possible to estimate the risk of exposure to plant PIs and the sensitivity of the aphid parasitoid A. ervi to these entomotoxins, by combining immunological, biochemical and biological approaches. First it pointed out that only immature stages are affected by PIs. Secondly, it documented two different modes of effect, according to the nature of the PIs and both host and parasitoid susceptibility. OCI prevented the development of A. ervi mainly due to the host susceptibility, whereas SbBBI only induced sublethal effects on the parasitoid, possibly due to both direct action on the parasitoid susceptible proteases, and host-mediated action through size reduction.     

Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethyl-amino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.     

Interaction of the fusogenic peptide B18 in its amyloid-state with lipid membranes studied by solid state NMR

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.     

Fgf signaling is required for zebrafish tooth development

We have investigated fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. We found that the tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocked dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remained intact as judged by normal pitx2 expression, not only was the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 was as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. Our results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme.     

Zebrafish topped is required for ventral motor axon guidance

Zebrafish primary motor axons extend along stereotyped pathways innervating distinct regions of the developing myotome. During development, these axons make stereotyped projections to ventral and dorsal myotome regions. Caudal primary motoneurons, CaPs, pioneer axon outgrowth along ventral myotomes; whereas, middle primary motoneurons, MiPs, extend axons along dorsal myotomes. Although the development and axon outgrowth of these motoneurons has been characterized, cues that determine whether axons will grow dorsally or ventrally have not been identified. The topped mutant was previously isolated in a genetic screen designed to uncover mutations that disrupt primary motor axon guidance. CaP axons in topped mutants fail to enter the ventral myotome at the proper time, stalling at the nascent horizontal myoseptum, which demarcates dorsal from ventral axial muscle. Later developing secondary motor nerves are also delayed in entering the ventral myotome whereas all other axons examined, including dorsally projecting MiP motor axons, are unaffected in topped mutants. Genetic mosaic analysis indicates that Topped function is non-cell autonomous for motoneurons, and when wild-type cells are transplanted into topped mutant embryos, ventromedial fast muscle are the only cell type able to rescue the CaP axon defect. These data suggest that Topped functions in the ventromedial fast muscle and is essential for motor axon outgrowth into the ventral myotome.     

Interactions among moths, crossbills, squirrels, and lodgepole pine in a geographic selection mosaic

Repeated patterns among biological communities suggest similar evolutionary and ecological forces are acting on the communities. Conversely, the lack of such patterns suggests that similar forces are absent or additional ones are present. Coevolution between a seed predator, the red crossbill (Loxia curvirostra complex), and lodgepole pine (Pinus contorta var. latifolia) exemplifies the ecological and evolutionary predictions for coevolving systems. In the absence of another seed predator and preemptive competitor (pine squirrels Tamiasciurus hudsonicus), natural selection by crossbills results in the evolution of larger cones with thicker distal scales, while relaxation of selection by squirrels results in the evolution of cones with more seeds and a greater ratio of seed mass to cone mass. However, in one range, the Little Rocky Mountains, distal scale thickness has diverged as expected but cone size has not. In these mountains seed predation by lodgepole pine cone borer moths (Eucosma recissoriana) was about 10 times greater than in other ranges lacking squirrels. We quantified moth predation and cone traits and found that moths select for smaller cones with fewer seeds. Thus, selection by moths in the Little Rocky Mountains counters both selection by crossbills for large cone size and relaxation of selection by squirrels favoring more seeds per cone and accounts for the relatively small and few-seeded cones in these mountains. It is also apparent that selection by crossbills changes seed defenses in a manner that favors seed predation by moths, whereas selection by squirrels likely reduces such predation. These results demonstrate the importance of considering the evolutionary consequences of community context in locally evolved (coevolved) traits and interactions.     

The significance of focal myoepithelial cell layer disruptions in human breast tumor invasion: a paradigm shift from the "protease-centered" hypothesis

Human breast epithelium and the stroma are separated by a layer of myoepithelial (ME) cells and basement membrane, whose disruption is a prerequisite for tumor invasion. The dissolution of the basement membrane is traditionally attributed primarily to an over-production of proteolytic enzymes by the tumor or the surrounding stromal cells. The results from matrix metalloproteinase inhibitor clinical trials, however, suggest that this "protease-centered" hypothesis is inadequate to completely reflect the molecular mechanisms of tumor invasion. The causes and signs of ME cell layer disruption are currently under-explored. Our studies revealed that a subset of pre- and micro-invasive tumors contained focal disruptions in the ME cell layers. These disruptions were associated with immunohistochemical and genetic alterations in the overlying tumor cells, including the loss of estrogen receptor expression, a higher frequency of loss of heterozygosity, and a higher expression of cell cycle, angiogenesis, and invasion-related genes. Focal ME layer disruptions were also associated with a higher rate of epithelial proliferation and leukocyte infiltration. We propose the novel hypothesis that a localized death of ME cells and immunoreactions that accompany an external environmental insult or internal genetic alterations are triggering factors for ME layer disruptions, basement membrane degradation, and subsequent tumor progression and invasion.     

Versatility of pyridine-2-methanol as a chelating ligand toward a manganese ion: synthesis and X-ray structural analysis on some manganese-pyridine-2-methanol derivatives

A systematic synthesis and X-ray structural analysis have been made for several manganese derivatives with pyridine-2-methanol as a chelating ligand; neutral Mn(C₅NH₄-2-CH₂OH)₂(C₆F₅CO₂)₂ (1), trans-[Mn(C₅H₄N-2-CH₂-OH)₂{C₆F₄-1,4-(CO₂)₂}]_∞ (2), cis-[Mn(C₅H₄N-2-CH₂-OH)₂{C₆F₄-1,3-(CO₂)₂}]_∞ (3), {Mn(C₅H₄N-2-CH₂-OH)₂(4,4^′-bipyridine)(ClO₄)}_∞ (4), and Mn(C₅H₄N-2-CH₂-OH)₃(ClO₄)₂(4,4-azopiridine) (pyridine-2-methanol) (5) are our results. 1 and 5 are monomers, while 2-4 are polymers. An oxidation state of the manganese ion in 1, 2, 3, and 5 is 2⁺, while that of 4 is suggested to be 3⁺. The magnetic data of 4 down to 2 K are measured. The length of the linker ligand has been suggested to afford a crucial effect on the dimensionality of the product.