DNA methylation variability at growth-related imprints does not contribute to overweight in monozygotic twins discordant for BMI

Defective genomic imprinting is often associated with syndromes that include abnormal growth as a clinical phenotype. However, whether differential methylation at imprinted loci also contributes to nonsyndromic abnormal body weight regulation is yet unknown. In this study, we investigated a potential contribution of aberrant DNA methylation at nine differentially methylated regions (DMRs) to the development of nonsyndromic overweight. Sixteen monozygotic (MZ) twins discordant for BMI (BMI difference ranging from 2.9-9.5 kg/m(2)) were recruited from the East Flanders Prospective Twin Survey. DNA extracted from saliva samples was bisulfite-treated followed by PCR amplification of target regions in DMRs most representative for abnormal growth syndromes: KvDMR1, H19 CTCF4, H19 CTCF6, IGF2 DMR0, IGF2 DMR2, GRB10, MEST, SNRPN, GNAS XL-α-s and GNAS Exon1A. At the DMRs analyzed, methylation-dependent primer extension experiments revealed only small intrapair differences in methylation indexes (MI) between the heavy and lean co-twins. In addition, no significant correlations between intrapair BMI differences and intrapair differences in MI were observed. In conclusion, DNA methylation variability at the nine DMRs analyzed does not seem to contribute to the discordancy in BMI observed in these MZ twins.     

Genotyping of Chromobacterium violaceum isolates by recA PCR-RFLP analysis

Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.     

Complex formation between the UL16 and UL21 tegument proteins of pseudorabies virus

The products of the UL16 and UL21 genes represent tegument proteins which are conserved throughout the mammalian herpesviruses. To identify and functionally characterize the respective proteins in the alphaherpesvirus pseudorabies virus, monospecific antisera against bacterially expressed fusion proteins were generated. In immunoblots the UL16 antiserum detected a ca. 40-kDa protein in infected cells and purified virion preparations, whereas the anti-UL21 serum recognized a protein of approximately 60 kDa. Interestingly, in immunoprecipitations using either antiserum, both proteins were coprecipitated, demonstrating the formation of a physical complex. To investigate protein function, viruses lacking either UL16, UL21, or both were constructed. Mutant viruses could be propagated on noncomplementing cells, indicating that these proteins, either alone or in combination, are not required for viral replication in cell culture. However, plaque sizes and viral titers were reduced. Electron microscopy showed only slight alterations in cytoplasmic virion morphogenesis, whereas intranuclear maturation stages were not affected. Similar results were obtained with a triple mutant simultaneously lacking the three conserved tegument proteins UL11, UL16, and UL21. In summary, our results uncover a novel interaction between conserved herpesvirus tegument proteins that increases the complexity of the intricate network of protein-protein interactions involved in herpesvirus morphogenesis.     

Micropropagation of squill (Charybdis numidica) through nodule culture

A micropropagation protocol for squill (Charybdis numidica, Hyacinthaceae) was developed using nodule culture. Nodule formation on leaf sections was induced in liquid Murashige and Skoog (MS) medium supplemented with 20 microM N6-benzylaminopurine (BA) under dark conditions. Nodules were cultured on semi-solid MS medium with factorial combinations of BA (0-40 microM) and alpha-naphthaleneacetic acid (NAA) (0-10 microM) under continuous light. Shoot regeneration from nodules occurred at varying degrees on all media. The highest number of shoots was formed on medium containing 2.5 microM NAA and 20 microM BA, while the maximum number of regenerated bulblets per gram nodule was induced on culture medium supplemented with 2.5 microM NAA alone. Regenerated shoots were successfully rooted at approximately 92% on semi-solid MS medium supplemented with 10 microM indole-3-acetic acid (IAA). Plantlets could be hardened and grew well after transfer to the greenhouse. Chemical analyses showed consistent bufadienolide patterns from cloned plantlets and the mother plant.     

Inferring the effects of demography and selection on Drosophila melanogaster populations from a chromosome-wide scan of DNA variation

Identifying regions of the Drosophila melanogaster genome that have been recent targets of positive Darwinian selection will provide evidence for adaptations that have helped this species to colonize temperate habitats. We have begun a search for such genomic regions by analyzing multiple loci (about 250) dispersed across the X chromosome in a putatively ancestral population from East Africa and a derived European population. For both populations we found evidence for past changes in population size. We estimated that a major bottleneck associated with the colonization of Europe occurred about 3,500-16,000 years ago. We also found that while this bottleneck can account for most of the reduction in variation observed in the European sample, there is a deficit of polymorphism in some genomic regions that cannot be explained by demography alone.     

Ascent exhalations of Antarctic fur seals: a behavioural adaptation for breath-hold diving?

Novel observations collected from video, acoustic and conductivity sensors showed that Antarctic fur seals consistently exhale during the last 50-85% of ascent from all dives (10-160 m, n > 8000 dives from 50 seals). The depth of initial bubble emission was best predicted by maximum dive depth, suggesting an underlying physical mechanism. Bubble sound intensity recorded from one seal followed predictions of a simple model based on venting expanding lung air with decreasing pressure. Comparison of air release between dives, together with lack of variation in intensity of thrusting movement during initial descent regardless of ultimate dive depth, suggested that inhaled diving lung volume was constant for all dives. The thrusting intensity in the final phase of ascent was greater for dives in which ascent exhalation began at a greater depth, suggesting an energetic cost to this behaviour, probably as a result of loss of buoyancy from reduced lung volume. These results suggest that fur seals descend with full lung air stores, and thus face the physiological consequences of pressure at depth. We suggest that these regular and predictable ascent exhalations could function to reduce the potential for a precipitous drop in blood oxygen that would result in shallow-water blackout.     

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.