Dynamic expression of a LEF‐EGFP Wnt reporter in mouse development and cancer

We have characterized a transgenic mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of multimerized LEF‐1 responsive elements. In embryos, EGFP was detected in known sites of Wnt activation, including the primitive streak, mesoderm, neural tube, somites, heart, limb buds, mammary placodes, and whisker follicles. In vitro cultured transgenic embryonic fibroblasts upregulated EGFP expression in response to activation of Wnt signaling by GSK3β inhibition. Mammary tumor cell lines derived from female LEF‐EGFP transgenic mice treated with the carcinogen 7, 12‐dimethylbenz[a]anthracene (DMBA) also express EGFP. Thus, this transgenic line is useful for ex vivo and in vitro studies of Wnt signaling in development and cancer. genesis 48:183–194, 2010. © 2010 Wiley‐Liss, Inc.     

Ecological fits, mis-fits and lotteries involving insect herbivores on the invasive plant, Bunias orientalis

Exotic plants bring with them traits that evolved elsewhere into their new ranges. These traits may make them unattractive or even toxic to native herbivores, or vice versa. Here, interactions between two species of specialist (Pieris rapae and P. brassicae) and two species of generalist (Spodoptera exigua and Mamestra brassicae) insect herbivores were examined on two native crucifer species in the Netherlands, Brassica nigra and Sinapis arvensis, and an exotic, Bunias orientalis. Bu. orientalis originates in eastern Europe and western Asia but is now an invasive pest in many countries in central Europe. P. rapae, P. brassicae and S. exigua performed very poorly on Bu. orientalis, with close to 100% of larvae failing to pupate, whereas survival was much higher on the native plants. In choice experiments, the pierid butterflies preferred to oviposit on the native plants. Alternatively, M. brassicae developed very poorly on the native plants but thrived on Bu. orientalis. Further assays with a German Bu. orientalis population also showed that several specialist and generalist herbivores performed very poorly on this plant, with the exception of Spodoptera littoralis and M. brassicae. Bu. orientalis produced higher levels of secondary plant compounds (glucosinolates) than B. nigra but not S. arvensis but these do not appear to be important factors for herbivore development. Our results suggest that Bu. orientalis is a potential demographic ‘trap’ for some herbivores, such as pierid butterflies. However, through the effects of an evolutionary ‘lottery’, M. brassicae has found its way through the plant’s chemical ‘minefield’.     

Transformation of Quercus petraea litter: successive changes in litter chemistry are reflected in differential enzyme activity and changes in the microbial community composition

The links among the changes in litter chemistry, the activity of extracellular enzymes and the microbial community composition were observed in Quercus petraea litter. Three phases of decomposition could be distinguished. In the early 4-month stage, with high activities of β-glucosidase, β-xylosidase and cellobiohydrolase, 16.4% of litter was decomposed. Hemicelluloses were rapidly removed while cellulose and lignin degradation was slow. In months 4-12, with high endocellulase and endoxylanase activities, decomposition of cellulose prevailed and 31.8% of litter mass was lost. After the third phase of decomposition until month 24 with high activity of ligninolytic enzymes, the litter mass loss reached 67.9%. After 2 years of decay, cellulose decomposition was almost complete and most of the remaining polysaccharides were in the form of hemicelluloses. Fungi largely dominated over bacteria as leaf endophytes and also in the litter immediately before contact with soil, and this fungal dominance lasted until month 4. Bacterial biomass (measured as phospholipid fatty acid content) in litter increased with time but also changed qualitatively, showing an increasing number of Actinobacteria. This paper shows that the dynamics of decomposition of individual litter components changes with time in accordance with the changes in the microbial community composition and its production of extracellular enzymes.     

Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes

Lipoteichoic acid (LTA) is an important cell wall polymer in Gram-positive bacteria. The enzyme responsible for polyglycerolphosphate LTA synthesis is LtaS, first described in Staphylococcus aureus. Four LtaS orthologues, LtaS(BS) , YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay, we determined that all four proteins are Mn(2+) -dependent metal enzymes that use phosphatidylglycerol as a substrate. We show that LtaS(BS) , YfnI and YqgS can produce polymers, suggesting that these three proteins are bona-fide LTA synthases while YvgJ functions as an LTA primase, as indicated by the accumulation of a GroP-Glc(2) -DAG glycolipid. Western blot analysis of LTA produced by ltaS(BS) , yfnI, yqgS and yvgJ single, triple and the quadruple mutant, showed that LTA production was only abolished in the quadruple and the YvgJ-only expressing mutant. B. subtilis strains expressing YfnI in the absence of LtaS(BS) produced LTA of retarded mobility, presumably caused by an increase in chain length as suggested by a structural analysis of purified LTA. Taken together, the presented results indicate that the mere presence or absence of LTA cannot account for cell division and sporulation defects observed in the absence of individual enzymes and revealed an unexpected enzymatic interdependency of LtaS-type proteins in B. subtilis.     

Population admixture,biological invasions and the balance between local adaptation and inbreeding depression

When previously isolated populations meet and mix, the resulting admixed population can benefit from several genetic advantages, including increased genetic variation, the creation of novel genotypes and the masking of deleterious mutations. These admixture benefits are thought to play an important role in biological invasions. In contrast, populations in their native range often remain differentiated and frequently suffer from inbreeding depression owing to isolation. While the advantages of admixture are evident for introduced populations that experienced recent bottlenecks or that face novel selection pressures, it is less obvious why native range populations do not similarly benefit from admixture. Here we argue that a temporary loss of local adaptation in recent invaders fundamentally alters the fitness consequences of admixture. In native populations, selection against dilution of the locally adapted gene pool inhibits unconstrained admixture and reinforces population isolation, with some level of inbreeding depression as an expected consequence. We show that admixture is selected against despite significant inbreeding depression because the benefits of local adaptation are greater than the cost of inbreeding. In contrast, introduced populations that have not yet established a pattern of local adaptation can freely reap the benefits of admixture. There can be strong selection for admixture because it instantly lifts the inbreeding depression that had built up in isolated parental populations. Recent work in Silene suggests that reduced inbreeding depression associated with post-introduction admixture may contribute to enhanced fitness of invasive populations. We hypothesize that in locally adapted populations, the benefits of local adaptation are balanced against an inbreeding cost that could develop in part owing to the isolating effect of local adaptation itself. The inbreeding cost can be revealed in admixing populations during recent invasions.     

Attract and deter: a dual role for pyrrolizidine alkaloids in plant–insect interactions

Pyrrolizidine alkaloids (PAs) are the major defense compounds of plants in the Senecio genus. Here I will review the effects of PAs in Senecio on the preference and performance of specialist and generalist insect herbivores. Specialist herbivores have evolved adaptation to PAs in their host plant. They can use the alkaloids as cue to find their host plant and often they sequester PAs for their own defense against predators. Generalists, on the other hand, can be deterred by PAs. PAs can also affect survival of generalist herbivores. Usually generalist insects avoid feeding on young Senecio leaves, which contain a high concentration of alkaloids. Structurally related PAs can differ in their effects on insect herbivores, some are more toxic than others. The differences in effects of PAs on specialist and generalists could lead to opposing selection on PAs, which may maintain the genetic diversity in PA concentration and composition in Senecio species.     

Invitation to Metabomeeting 2011

Announcement

Invitation to Metabomeeting 2011 September 25–28, Helsinki, Finland     

Efficient Transfection of Novel Bovine Papillomavirus 1 Expression Plasmids

A series of novel bovine papillomavirus type 1 (BPV-1)-based expression plasmids was constructed and characterized in vitro as a starting point for the development of an in vivo gene therapeutic method. The order of transfection efficiency for different pBPVlacZ plasmids was pCGalBPV > pTKBPV > pSRalphaBPV in CV1-P cells. In the absence of selection pressure, the expression of pCGalBPVlacZ and pTKBPVlacZ was associated with long-term maintenance. In a comparison of pBPVlacZ with pSVlacZ, expression was maintained up to 12-17 and 8-12 days, respectively. The transfection of pBPVlacZ plasmids was efficient in secondary and primary, dividing and nondividing, neural and nonneural, and human cells and, furthermore, independent of the cell cycle as seen in growing as well as resting cells. All these characteristics are likely to be relevant for in vivo conditions, under which the percentage of proliferating cells could be quite low. In conclusion, the pBPV plasmids were efficiently delivered and expressed in different host cells, and therefore their performance in gene therapy is worth testing.     

Effects of TGF-beta 1 on interleukin profile of human dental pulp and odontoblasts

Transforming growth factor-beta 1 (TGF-beta1) is the most extensively studied growth factor in dentin-pulp complex, with pleiotropic effects on pulp response and healing. Our main objective was to analyze the expression profile of pulp tissue and odontoblasts, and the effects of TGF-beta1 on these profiles in cultured human pulp and odontoblasts with a specific interest in the anti- and pro-inflammatory cytokines. For that purpose, pulps and odontoblasts were cultured for different time periods, and microarray was performed to both cultured and native samples. Of cytokines, various interleukins (IL) were confirmed by RT-PCR, and in +/- TGF-beta1 treated pulps also by antibody array. Pro-inflammatory IL-7, -12alpha and -16 mRNAs were detected in native pulp. The expression levels of pro-inflammatory IL-1alpha, -1beta, -6 and -8 were clearly induced after TGF-beta1 treatment, while no anti-inflammatory cytokines were induced. Of all pulpal interleukins analyzed IL-6 and -8 were present at the highest levels in conditioned pulp tissue media. In native odontoblasts pro-inflammatory IL-6 and -7 mRNAs were detected, and in cultured odontoblasts pro-inflammatory IL-8 mRNA showed over 20-fold transient induction after TGF-beta1 treatment. Our results demonstrate that TGF-beta1 is a potent regulator of pro-inflammatory responses and defensive reactions in dentin-pulp complex.