Effect of summer drought relief on the impact of the root weevil Cyphocleonus achates on spotted knapweed

A recent decline in spotted knapweed, Centaurea stoebe L. subsp. micranthos (Asteraceae), has been observed in parts of western Montana. The release of the biological control agent Cyphocleonus achates (Fahraeus) is thought to contribute to the decline, but persistent drought since at least 1999 may be an additional factor. We conducted outdoor plot experiments to test the relative impacts of C. achates weevils and summer drought relief on spotted knapweed survival and growth. Groups of spotted knapweed transplants were assigned to one of four weekly water addition treatments (no added water, and 0.25, 0.5 or full recovery of plant water deficit, where "deficit" refers to potential evapotranspiration minus rainfall) in May to August 2004 and June to August 2005 and to either exposure to or protection from C. achates. In June of each subsequent year (2005 and 2006), plants were harvested and growth attributes that reflect plant vigor were measured. Drought indices showed that throughout the time of the study until January 2006, western Montana was in drought alert or severe drought. Summer drought relief had no effect on aboveground biomass and plant height of knapweed plants in subsequent years, but feeding by C. achates larvae reduced these two measures of plant vigor. Knapweed plants resuming growth after the drought ended in spring 2006 were significantly larger than those resuming growth under drought conditions in spring 2005. Spring drought may reduce knapweed growth, but C. achates reduced knapweed growth regardless of drought conditions.     

Analysis of compensatory beta-cell response in mice with combined mutations of Insr and Irs2

Type 2 diabetes results from impaired insulin action and beta-cell dysfunction. There are at least two components to beta-cell dysfunction: impaired insulin secretion and decreased beta-cell mass. To analyze how these two variables contribute to the progressive deterioration of metabolic control seen in diabetes, we asked whether mice with impaired beta-cell growth due to Irs2 ablation would be able to mount a compensatory response in the background of insulin resistance caused by Insr haploinsufficiency. As previously reported, approximately 70% of mice with combined Insr and Irs2 mutations developed diabetes as a consequence of markedly decreased beta-cell mass. In the initial phases of the disease, we observed a robust increase in circulating insulin levels, even as beta-cell mass gradually declined, indicating that replication-defective beta-cells compensate for insulin resistance by increasing insulin secretion. These data provide further evidence for a heterogeneous beta-cell response to insulin resistance, in which compensation can be temporarily achieved by increasing function when mass is limited. The eventual failure of compensatory insulin secretion suggests that a comprehensive treatment of beta-cell dysfunction in type 2 diabetes should positively affect both aspects of beta-cell physiology.     

Bim and Bcl-2 mutually affect the expression of the other in T cells

The life and death of T cells is controlled to a large extent by the relative amounts of Bcl-2-related proteins they contain. The antiapoptotic protein Bcl-2 and the proapoptotic protein Bim are particularly important in this process with the amount of Bcl-2 per cell dropping by about one-half when T cells prepare to die. In this study we show that Bcl-2 and Bim each control the expression of the other. Absence of Bim leads to a drop in the amount of intracellular Bcl-2 protein, while having no effect on the amounts of mRNA for Bcl-2. Conversely, high amounts of Bcl-2 per cell allow high amounts of Bim, although in this case the effect involves increases in Bim mRNA. These mutual effects occur even if Bcl-2 is induced acutely. Thus these two proteins control the expression of the other, at either the protein or mRNA level.     

Biological and molecular studies of a cypovirus from the black fly Simulium ubiquitum (Diptera: Simuliidae)

A cypovirus from the black fly Simulium ubiquitum (SuCPV) was isolated and examined using biological and molecular techniques. SuCPV produces small (typically 0.25mum), polyhedral shaped inclusion bodies (polyhedra), in which the virus particles become multiply embedded. SuCPV is the third cypovirus isolated from Diptera, but the first from Simuliidae that has been characterized using molecular analyses. SuCPV has a genome composed of 10 segments of dsRNA, with an electrophoretic migration pattern that is different from those of recent UsCPV-17 and CrCPV-17 isolates from the mosquitoes Uranotaenia sapphirina and Culex restuans, respectively. The SuCPV electropherotype appears to show significant differences from those of the previously characterized lepidopteran cypoviruses. Sequence analysis of SuCPV segment 10 shows that it is unrelated to either of the two CPV isolates from Diptera or to the CPV species for which Seg-10 has been previously characterized from Lepidoptera. A comparison of the terminal regions of SuCPV genome segments to those of CPV-1, 2, 4, 5 14, 15, 16, 17, 18, and 19 also revealed only low levels of conservation. We therefore, propose that SuCPV is classified within a new Cypovirus species, which we have tentatively identified as Cypovirus-20. We have therefore referred to this virus isolate as S. ubiquitum CPV-20 (SuCPV-20).     

Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract

Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia in infants and children. Currently, palivizumab is the only approved monoclonal antibody (mAb) for prophylaxis of RSV. However, a small percentage of patients are not protected by palivizumab; in addition, palivizumab does not inhibit RSV replication effectively in the upper respiratory tract. We report here the development and characterization of motavizumab, an ultra-potent, affinity-matured, humanized mAb derived from palivizumab. Several palivizumab variants that enhanced the neutralization of RSV in vitro by up to 44-fold were generated; however, in vivo prophylaxis of cotton rats with these antibodies conferred only about a twofold improvement in potency over palivizumab. This unexpected small increase of in vivo potency was caused by poor serum pharmacokinetics and lung bio-availability that resulted from unexpectedly broad tissue binding. Subsequent analyses revealed that changes at three amino acids arising from the affinity maturation markedly increased the non-specific binding to various tissues. Our results suggested that k(on)-driven mutations are more likely to initiate non-specific binding events than k(off)-driven mutations. Reversion of these three residues to the original sequences greatly diminished the tissue binding. The resulting mAb, motavizumab, binds to RSV F protein 70-fold better than palivizumab, and exhibits about a 20-fold improvement in neutralization of RSV in vitro. In cotton rats, at equivalent concentrations, motavizumab reduced pulmonary RSV titers to up to 100-fold lower levels than did palivizumab and, unlike palivizumab, motavizumab very potently inhibited viral replication in the upper respiratory tract. This affinity-enhanced mAb is being investigated in pivotal clinical trials. Importantly, our engineering process offers precious insights into the improvement of other therapeutic mAbs.     

Inferring developmental constraint and constraint release: primordial germ cell determination mechanisms as examples

Developmental constraint and its converse constraint release are significant concepts in understanding pattern and process in macroevolution. The purpose of this paper is to propose a two-step method for identifying constraints and constraint release. The first step is a phylogenetic optimization procedure to identify which trait/process is primitive and which is derived. The primitive trait is inferred to be the constraint and the convergently derived trait the release. The second criterion uses sister-clade asymmetry. Clades diagnosed by the constraint will have fewer taxa than clades diagnosed by the release. As an example, we use the process of germ cell specification, in which there are three modes of specification. Our results corroborate previous conclusions that the induced mode is the constraint and the predetermined mode is the release and we speculate on the importance of these two processes in terms of robustness and evolvability.     

Substitution of tyrosine 146 in the dI component of proton-translocating transhydrogenase leads to reversible dissociation of the active dimer into inactive monomers

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The protein has three components: dI binds NADH, dIII binds NADP+, and dII spans the membrane. Transhydrogenase is a "dimer" of two dI-dII-dIII "monomers"; x-ray structures suggested that the two catalytic sites alternate during turnover. Invariant Tyr146 in recombinant dI of Rhodospirillum rubrum transhydrogenase was substituted with Phe and Ala (proteins designated dI.Y146F and dI.Y146A, respectively). Analytical ultracentrifuge experiments and differential scanning calorimetry show that dI.Y146A more readily dissociates into monomers than wild-type dI. Analytical ultracentrifuge and Trp fluorescence experiments indicate that the dI.Y146A monomers bind NADH much more weakly than dimers. Wild-type dI and dI.Y146F reconstituted activity to dI-depleted membranes with similar characteristics. However, dI.Y146A reconstituted activity in its dimeric form but not in its monomeric form, this despite monomers retaining their native fold and binding to the dI-depleted membranes. It is suggested that transhydrogenase reconstructed with monomers of dI.Y146A is catalytically compromised, at least partly as a consequence of the lowered affinity for NADH, and this results from lost interactions between the nucleotide binding site and the protein beta-hairpin upon dissociation of the dI dimer. The importance of these interactions and their coupling to dI domain rotation in the mechanism of action of transhydrogenase is emphasized. Two peaks in the 1H NMR spectrum of wild-type dI are broadened in dI.Y146A and are tentatively assigned to S-methyl groups of Met resonances in the beta-hairpin, consistent with the segmental mobility of this feature in the structure.     

alpha v beta3 and alpha5beta1 integrin recycling pathways dictate downstream Rho kinase signaling to regulate persistent cell migration

Accumulating evidence suggests that integrin recycling regulates cell migration. However, the lack of reagents to selectively target the trafficking of individual heterodimers, as opposed to endocytic transport as a whole, has made it difficult to define the contribution made by particular recycling pathways to directional cell movement. We show that autophosphorylation of protein kinase D1 (PKD1) at Ser(916) is necessary for its association with alphavbeta3 integrin. Expression of PKD1(916A) or the use of mutants of beta3 that do not bind to PKD1 selectively inhibits short-loop, Rab4-dependent recycling of alphavbeta3, and this suppresses the persistence of fibroblast migration. However, we report that short-loop recycling does not directly contribute to fibroblast migration by moving alphavbeta3 to the cell front, but by antagonizing alpha5beta1 recycling, which, in turn, influences the cell's decision to migrate with persistence or to move randomly.     

Knockdown and mortality comparisons among spinosad-, imidacloprid-, and methomyl-containing baits against susceptible Musca domestica (Diptera: Muscidae) under laboratory conditions

The activity of spinosad, imidacloprid, and methomyl baits and technical actives were assessed against susceptible house flies, Musca domestica L. (Diptera: Muscidae). In a feeding assay, imidacloprid affected flies more rapidly than methomyl or spinosad, but spinosad was 2.7 times more potent than methomyl and 8 times more potent than imidacloprid. The profile of technical actives correlated with their respective fly bait formulations in laboratory assays. Although having the most rapid onset of activity in laboratory tests, up to 50% of flies remained alive after exposure to imidacloprid bait. In contrast, <5% of flies survived 24-h exposure to spinosad or methomyl baits. High temperature reduced the knockdown activity of imidacloprid bait and slowed the speed of kill for spinosad and methomyl baits over a 24-h exposure period. Spinosad and methomyl baits were also superior to imidacloprid when applied to the floors of environmentally controlled rooms at label recommended rates, providing good fly control for up to 21 d. The fact that a significant percentage of flies exposed to imidacloprid were rapidly knocked down but subsequently remained alive in all of the assays suggested that flies were recovering from initial exposure to this compound. Given its favorable safety profile, a high degree of initial and residual activity comparable with methomyl and lack of cross-resistance to other chemistries, spinosad bait may be a valuable component of house fly control programs to help control or delay the emergence of resistant populations.