Intramolecular masking of nuclear localization signals: analysis of importin binding using a novel AlphaScreen-based method

Active nuclear import of proteins requires the recognition of a nuclear localization sequence (NLS) by members of the importin (IMP) family of proteins. We have developed a modified AlphaScreen-based assay able to estimate the solution binding affinities of such interactions using biotinylated IMPs and His6-tagged NLS-containing proteins. We describe this assay in detail as well as its application in documenting the phenomenon of intramolecular masking of NLSs using recombinant green fluorescent protein (GFP) fusion proteins containing sequences from the SV40 large tumor T antigen (T-ag). We also use it to examine, for the first time, IMP binding to the cancer cell-specific proapoptotic factor viral protein 3 (VP3) from the chicken anemia virus (CAV). High-affinity binding of the IMPalpha/beta heterodimer to the T-ag NLS was observed when the GFP tag was fused to its N terminus but not to its C terminus. Effects of flanking residues were also observed in GFP-T-ag fusion derivatives containing the Thr128 NLS-inactivating mutation, whereby the absence of flanking sequences N terminal to the T-ag NLS appeared to decrease the specificity of the mutation in terms of oblating IMPalpha/beta binding. IMPbeta, but not IMPalpha or the IMPalpha/beta heterodimer, was found to bind to CAV VP3 with high affinity. Interestingly, GFP-VP3(74-121) bound to IMPbeta with threefold higher affinity than the full-length protein, GFP-VP3(1-121), implying that the NLS is masked to a significant extent in the context of full-length protein. This may represent a regulatory mechanism to control nuclear import in a tumor cell-specific fashion.     

Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability

Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.     

Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.     

Flowering phenology and sexual allocation in single-mutation lineages of Arabidopsis thaliana

We investigated the relationship between flowering time and sexual allocation in wild-type Arabidopsis thaliana and in genetically similar lineages with single-locus mutations of floral induction genes. We examined whether the mechanisms of growth and development that govern resource investment would permit the independent evolution of reproductive phenology and sexual allocation, or whether constraints, manifested as pleiotropic effects of the single mutations, would link these two life-history traits. Flowering times differed significantly among genotypes, and, as expected, later flowering times were associated with larger vegetative size. Later flowering genotypes produced heavier floral parts (larger petals, in particular), and allocated a significantly lower proportion of biomass to androecia, especially in final allocations that included fruit biomass. At least part of this pleiotropic covariation of flowering time and sexual allocation is likely to be mediated by vegetative size and the rate of resource supply to growing reproductive tissues, because the larger fruits of late-flowering genotypes required the same time, or proportionately less time than the difference in biomass, to mature. Because fruit mass is considered an investment in female function, sexual allocation measured at the end of a growing season tends to be highly female biased in angiosperms. We consider the implications of the pleiotropic association of flowering time, vegetative size, and sexual investment for the theory of sex allocation, and suggest that the idiosyncratic phenology of sexual investment in flowering plants creates a departure from a central assumption of Fisher's seminal sex allocation argument.     

Independent life history evolution between generations of bivoltine species: a case study of cyclical parthenogenesis

Successive generations of bi- and multivoltine species encounter differing biotic and abiotic environments intra-annually. The question of whether selection can independently adjust the relationship between body size and components of reproductive effort within successive generations in response to generation-specific environmental variation is applicable to a diversity of taxa. Herein, we develop a conceptual framework that illustrates increasingly independent life history adjustments between successive generations of taxa exhibiting complex life cycles. We apply this framework to the reproductive biology of the gall-forming insect, Belonocnema treatae (Hymenoptera: Cynipidae). This bivoltine species expresses cyclical parthenogenesis in which alternating sexual and asexual generations develop in different seasons and different environments. We tested the hypotheses that ecological divergence between the alternate generations is accompanied by generational differences in body size, egg size, and egg number and by changes in the relationships between body size and these components of reproductive effort. Increased potential reproductive effort of sexual generation B. treatae is attained by increased body size and egg number (with no trade-off between egg number and egg size) and by a significant increase in the slope of the relationship between body size and potential fecundity. These generation-specific relationships, interpreted in the context of the model framework, suggest that within each generation selection has independently molded the relationships relating body size to potential fecundity and potential reproductive effort in B. treatae. The conceptual framework is broadly applicable to comparisons involving the alternating generations of bi- and multivoltine species.     

Histories of host shifts and cospeciation among free‐living parasitoids of Rhagoletis flies

Host shifts by specialist insects can lead to reproductive isolation between insect populations that use different hosts, promoting diversification. When both a phytophagous insect and its ancestrally associated parasitoid shift to the same novel host plant, they may cospeciate. However, because adult parasitoids are free living, they can also colonize novel host insects and diversify independent of their ancestral host insect. Although shifts of parasitoids to new insect hosts have been documented in ecological time, the long‐term importance of such shifts to parasitoid diversity has not been evaluated. We used a genus of flies with a history of speciation via host shifting (Rhagoletis [Diptera: Tephritidae]) and three associated hymenopteran parasitoid genera (Diachasma, Coptera and Utetes) to examine cophylogenetic relationships between parasitoids and their host insects. We inferred phylogenies of Rhagoletis, Diachasma, Coptera and Utetes and used distance‐based cophylogenetic methods (ParaFit and PACo) to assess congruence between fly and parasitoid trees. We used an event‐based method with a free‐living parasitoid cost model to reconstruct cophylogenetic histories of each parasitoid genus and Rhagoletis. We found that the current species diversity and host–parasitoid associations between the Rhagoletis flies and parasitoids are the primary result of ancient cospeciation events. Parasitoid shifts to ancestrally unrelated hosts primarily occur near the branch tips, suggesting that host shifts contribute to recent parasitoid species diversity but that these lineages may not persist over longer time periods. Our analyses also stress the importance of biologically informed cost models when investigating the coevolutionary histories of hosts and free‐living parasitoids.     

Circular permutation of granulocyte colony-stimulating factor

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.     

Pretreatment with periodate-oxidized adenosine enhances developmental toxicity of inorganic arsenic in mice

BACKGROUND: Inorganic arsenic, given by injection to pregnant laboratory animals, can induce malformations. Arsenic methylation can be inhibited by periodate‐oxidized adenosine (PAD). Severe human health effects from high chronic arsenic exposure have mainly been reported in populations with significant levels of malnutrition, which may enhance toxicity by diminishing arsenic methylating capacity. This study sought to determine the effect of inhibition of arsenic methylation on the developmental toxicity of arsenic in a mammalian model. METHODS: PAD (100 µM/kg, i.p.), was given to pregnant CD‐1 strain mice 30min before 7.5mg/kg sodium arsenite [As(III)], i.p., or 17.9mg/kg sodium arsenate [As(V)], i.p., on gestation day 8 (GD 8; copulation plug=GD 0). Control dams received As(III), As(V), or PAD alone or were untreated. Test dams were killed on GD 17, and their litters were examined for mortality and gross and skeletal defects. RESULTS: Pretreatment with PAD before either arsenical resulted in increased maternal toxicity and lower fetal weights. Pretreatment also caused higher prenatal mortality, with 8 of 21 and 5 of 17 litters totally resorbed in the PAD plus As(III) and PAD plus As(V) treatment groups, respectively. Significant increases in the incidences of exencephaly, ablepharia, and anomalies of the vertebral centra, sternebrae, and ribs were also associated with PAD pretreatment. Short tail (3 fetuses in 3 litters) was seen only following PAD plus As(III) treatment. CONCLUSIONS: These results demonstrate that the developmental toxicity of inorganic arsenic can be enhanced by PAD, due possibly to inhibited methylation of arsenic. Birth Defects Res B 68:335–343, 2003. © 2003 Wiley‐Liss, Inc.     

Conversion of glucose, acetate and lactate to CO2 and fatty acids in liver and adipose tissue of prairie voles (Microtus ochrogaster)

Production of CO2 and fatty acids from acetate, glucose and lactate was determined in slices of liver and adipose tissue from prairie voles fed either a high-starch or a high-cellulose diet. Acetate and lactate were oxidized to CO2 and converted to fatty acids at greater rates than was glucose in both liver and adipose tissue. Fatty acid synthesis occurred at greater rates in adipose tissue than in liver. Fatty acid synthesis per adipocyte increased with increased adipocyte diameter. Fiber content of diets had only minimal effect on metabolic activities of liver and adipose tissue.