Modulation of Lck function through multisite docking to T cell-specific adapter protein

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr(280), Tyr(290), and Tyr(305) were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain. Binding affinities of the TSAd Tyr(P)(280) and Tyr(P)(290) phosphopeptides to the isolated Lck SH2 domain were similar to that observed for the Lck Tyr(P)(505) phosphopeptide, whereas the TSAd Tyr(P)(305) peptide displayed a 10-fold higher affinity. The proline-rich Lck SH3-binding site on TSAd as well as the Lck SH2 domain were required for efficient tyrosine phosphorylation of TSAd by Lck. Interaction sites on TSAd for both Lck SH2 and Lck SH3 were necessary for TSAd-mediated modulation of proximal TCR signaling events. We found that 20-30% of TSAd molecules are phosphorylated in activated T cells and that the proportion of TSAd to Lck molecules in such cells is approximately 1:1. Therefore, in activated T cells, a considerable number of Lck molecules may potentially be engaged by TSAd. In conclusion, Lck binds to TSAd prolines and phosphorylates and interacts with the three C-terminal TSAd tyrosines. We propose that through multivalent interactions with Lck, TSAd diverts Lck from phosphorylating other substrates, thus modulating its functional activity through substrate competition.     

Maternal and paternal contribution to intergenerational recurrence of breech delivery: population based cohort study

Objective To investigate intergenerational recurrence of breech delivery, with a hypothesis that both women and men delivered in breech presentation contribute to increased risk of breech delivery in their offspring.Design Population based cohort study for two generations.Setting Data from the medical birth registry of Norway, based on all births in Norway 1967-2004 (2.2 million births).Participants Generational data were provided through linkage by national identification numbers, forming 451 393 mother-offspring units and 295 253 father-offspring units. We included units where both parents and offspring were singletons and offspring were first born, forming 232 704 mother-offspring units and 154 851 father-offspring units for our analyses.Main outcome measure Breech delivery in the second generation.Results Men and women who themselves were delivered in breech presentation had more than twice the risk of breech delivery in their own first pregnancies compared with men and women who had been cephalic presentations (odds ratios 2.2, 95% confidence interval 1.8 to 2.7, and 2.2, 1.9 to 2.5, for men and women, respectively). The strongest risks of recurrence were found for vaginally delivered offspring and were equally strong for men and women. Increased risk of recurrence of breech delivery in offspring was present only for parents delivered at term.Conclusion Intergenerational recurrence risk of breech delivery in offspring was equally high when transmitted through fathers and mothers. It seems reasonable to attribute the observed pattern of familial predisposition to term breech delivery to genetic inheritance, predominantly through the fetus.     

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1? mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.     

Reproductive biology of the arctic collembolan Hypogastrura tullbergi

Terrestrial organisms of the Arctic are faced with strong climatic fluctuations. Predictable seasonality with cold/long winters and short/cool summers are combined with unpredictable between and within year variation. This indicates that various selection pressures act on the reproductive strategies of the populations. The arctic collembolan Hypogastrura tullbergi reproduces in a short period following snow melt. Hatching occurs in late summer, the animals grow to adult size within their second summer and reproduce for the first time in the beginning of their third summer. We performed several experiments to determine the reproductive investment and proximate mechanism that regulate timing and duration of reproduction. We found that H. tullbergi entered a reproductive diapause when reared at constant temperature, a diapause that was terminated by a cold exposure (winter). Surprisingly, cold exposure of small juveniles may also prevent development of a reproductive diapause in adults. Thus, the life-cycle normally spanning 2 yr can potentially be reduced to one year if the overwintering juveniles reach maturity before the end of the reproductive period in the field. After termination of the diapause, the animals reproduced up to 3 times during a period of 6 weeks at 15°C. This reproductive period was considerably longer (measured in degree days) than the one observed in the field. Our results suggested that temperature quiescence, i.e. the inability to reproduce under a certain temperature threshold, may adjust the termination of the reproductive period with current temperature before a new diapause occurs in late summer. The cost of reproduction was low and suggests that it can be adaptive to spread reproduction over more than one year. The results are discussed in relation to the arctic climate and strategies favoured by unpredictable and predictable (seasonal) variations in the environment. The present study forms part of a larger investigation on population dynamics and life history strategies of H. tullbergi from the Arctic.     

The Mitogen-activated protein kinase p38 links Shiga Toxin-dependent signaling and trafficking

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca(2+)-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca(2+) levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca(2+) levels. Intracellular transport of Stx is Ca(2+) dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca(2+) and p38, to regulate its trafficking to the Golgi apparatus.     

Genome dynamics in major bacterial pathogens

Pathogenic bacteria continuously encounter multiple forms of stress in their hostile environments, which leads to DNA damage. With the new insight into biology offered by genome sequences, the elucidation of the gene content encoding proteins provides clues toward understanding the microbial lifestyle related to habitat and niche. Campylobacter jejuni, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis , the pathogenic Neisseria, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus are major human pathogens causing detrimental morbidity and mortality at a global scale. An algorithm for the clustering of orthologs was established in order to identify whether orthologs of selected genes were present or absent in the genomes of the pathogenic bacteria under study. Based on the known genes for the various functions and their orthologs in selected pathogenic bacteria, an overview of the presence of the different types of genes was created. In this context, we focus on selected processes enabling genome dynamics in these particular pathogens, namely DNA repair, recombination and horizontal gene transfer. An understanding of the precise molecular functions of the enzymes participating in DNA metabolism and their importance in the maintenance of bacterial genome integrity has also, in recent years, indicated a future role for these enzymes as targets for therapeutic intervention.     

DNA repair in Mycobacterium tuberculosis revisited

Our understanding of Mycobacterium tuberculosis DNA repair mechanisms is still poor compared with that of other bacterial organisms. However, the publication of the first complete M. tuberculosis genome sequence 10 years ago boosted the study of DNA repair systems in this organism. A first step in the elucidation of M. tuberculosis DNA repair mechanisms was taken by Mizrahi and Andersen, who identified homologs of genes involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes required for nucleotide excision repair, base excision repair, recombination, and SOS repair and mutagenesis were identified. Notably, no homologs of genes involved in mismatch repair were identified. Novel characteristics of the M. tuberculosis DNA repair machinery have been found over the last decade, such as nonhomologous end joining, the presence of Mpg, ERCC3 and Hlr – proteins previously presumed to be produced exclusively in mammalian cells – and the recently discovered bifunctional dCTP deaminase:dUTPase. The study of these systems is important to develop therapeutic agents that can counteract M. tuberculosis evolutionary changes and to prevent adaptive events resulting in antibiotic resistance. This review summarizes our current understanding of the M. tuberculosis DNA repair system.     

Structure of the Malpighian tubule cells and annual changes in the structure and chemical composition of their spherites in the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae,Saltatoria)

Periodical changes in the structure of spherites in the Malpighian tubule cells of the cave cricket Troglophilus neglectus were studied to elucidate their role during the cricket's life cycle in natural circumstances. Special interest was given to the dormant overwintering period when we hypothesized that the primary role of spherites is to supply minerals for basic vital processes. The investigation was carried out by light and transmission electron microscopy, energy dispersive X-ray spectroscopy, electron energy-loss spectroscopy and energy-filtering TEM. Spherites are present only in the middle Malpighian tubule segment, consisting of Type 1 cells, characterized, among other features, by a round, apically placed nucleus and numerous spherites, and a few Type 2 cells with an elongated nucleus in the centre and sparse spherites. At the beginning of dormancy in November juveniles, minerals are accumulated in spherites and then decline until March. In one-year-old May larvae, spherites are commonly rich in minerals, and from July onwards they are progressively exploited in the adults. Spherite destruction starts with apoptosis in senile October individuals. The findings suggest that the mineral supply of spherites in Malpighian tubules is crucial to supporting vital processes throughout the life cycle of T. neglectus.     

Parasitism of the house fly parasitoid <Emphasis Type="Italic">Spalangia cameroni</Emphasis> on Norwegian pig farms: local effect of release method

Mass release of parasitoids (Hymentoptera: Pteromalidae) is one possible control method of house flies (Musca domestica L.) on livestock farms. To improve the success of this method, however, there is a need for more detailed recommendations. In the present study, parasitism was evaluated in and around pens following release of the parasitoid Spalangia cameroni Perkins by hand and from containers. The study was conducted at conventional Norwegian pig farms with scattered breeding grounds for house flies. The experiment was carried out twice, with a total of seven trials of each release method followed by four weeks of monitoring parasitism by house fly sentinel pupae. No significant difference was found between the two release methods. Parasitism decreased with temperature (range 18–23°C) and was low on farms with few sites for the parasitoids to hide.