The Saccharomyces cerevisiae type 2A protein phosphatase Pph22p is biochemically different from mammalian PP2A.

The Saccharomyces cerevisiae type 2A protein phosphatase (PP2A) Pph22p differs from the catalytic subunits of PP2A (PP2Ac) present in mammals, plants and Schizosaccharomyces pombe by a unique N-terminal extension of approximately 70 amino acids. We have overexpressed S. cerevisiae Pph22p and its N-terminal deletion mutant Delta N-Pph22p in the GS115 strain of Pichia pastoris and purified these enzymes to apparent homogeneity. Similar to other heterologous systems used to overexpress PP2Ac, a low yield of an active enzyme was obtained. The recombinant enzymes designed with an 8 x His-tag at their N-terminus were purified by ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on Ni2+-nitrilotriacetic acid agarose. Comparison of biochemical properties of purified Pph22p and Delta N-Pph22p with purified human 8 x His PP2Ac identified similarities and differences between these two enzymes. Both enzymes displayed similar specific activities with 32P-labelled phosphorylase a as substrate. Furthermore, selected inhibitors and metal ions affected their activities to the same extend. In contrast to the mammalian catalytic subunit PP2Ac, but similar to the dimeric form of mammalian PP2A, Pph22p, but not Delta N-Pph22p, interacted strongly with protamine. Also with regard to the effects of protamine and polylysine on phosphatase activity Pph22p, but not Delta N-Pph22p, behaved similarly to the PP2Ac-PR65 dimer, indicating a regulatory role for the N-terminal extension of Pph22p. The N-terminal extension appears also responsible for interactions with phospholipids. Additionally Pph22p has different redox properties than PP2Ac; in contrast to human PP2Ac it cannot be reactivated by reducing agents. These properties make the S. cerevisiae Pph22p phosphatase a unique enzyme among all type 2A protein phosphatases studied so far.     

Contribution of regulatory T cells and effector T cell deletion in tolerance induction by costimulation blockade

Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F(1) offspring (C57BL/6 x C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25(+) cell-depleted splenocyte transfer, showing that CD25(+) natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6-30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3(+) cells among donor CD4(+) cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance.     

Experimental evidence for a causal effect of pair-bond duration on reproductive performance in oystercatchers (Haematopus ostralegus)

Many studies have suggested that reproductive performance improvesduring the pair-bond, which might explain why individuals rematewith the same partner in many species. However, discussion existsabout whether the association between reproductive performanceand pair-bond duration that is reported in these studies reflectsa causal relationship. Usually it is unclear whether a positiveassociation is caused by pairs improving during their pair-bondor by high-quality pairs staying together for longer. Furthermore,reproductive performance often also depends on the age or breedingexperience of parents, which all covary with pair-bond duration.A much needed experimental approach is lacking so far. We investigatedthe effect of pair-bond duration on reproductive performancein a long-lived monogamous bird species based on natural aswell as experimental variation. The duration of oystercatcher(Haematopus ostralegus) pair-bonds, which were followed for21 years, strongly affected reproductive output, even aftercontrolling for effects of age and breeding experience. Pairsimproved during their pair-bond, and there were no indicationsof selective disappearance of low-quality pairs; however, pairsthat stayed together for very long performed badly. Experimentalremoval of one partner showed that the reproductive cost ofdivorce depended on the pair-bond duration with the old partner.In addition, after remating, the newly formed pairs stronglyimproved again, independent of the age and breeding experienceof the remated pair members. As such, this study provides thefirst experimental evidence of a causal effect of pair-bondduration on reproductive performance.     

Irreducible incoherence and intelligent design: a look into the conceptual toolbox of a pseudoscience

The concept of Irreducible Complexity (IC) has played a pivotal role in the resurgence of the creationist movement over the past two decades. Evolutionary biologists and philosophers have unambiguously rejected the purported demonstration of "intelligent design" in nature, but there have been several, apparently contradictory, lines of criticism. We argue that this is in fact due to Michael Behe's own incoherent definition and use of IC. This paper offers an analysis of several equivocations inherent in the concept of Irreducible Complexity and discusses the way in which advocates of the Intelligent Design Creationism (IDC) have conveniently turned IC into a moving target. An analysis of these rhetorical strategies helps us to understand why IC has gained such prominence in the IDC movement, and why, despite its complete lack of scientific merits, it has even convinced some knowledgeable persons of the impending demise of evolutionary theory.     

Strong but variable associations between social dominance and clutch sex ratio in a colonial corvid

We studied primary sex ratio of clutches in relation to socialdominance for 6 years in a colony of free-living jackdaws, asmall corvid. Social dominance was strongly associated withclutch sex ratio, with the difference in clutch sex ratio betweenthe most and least dominant pairs being 30–40%. To ourknowledge, this is the first demonstration of an associationbetween social dominance and sex allocation in birds. However,the direction of this effect varied between years. Dominantjackdaws produced more sons during the first years of the studybut fewer sons during the last years. Offspring sex was notrelated to laying order within a clutch, and the effect of socialdominance on sex ratio was similar on eggs laid first, middle,or last. We investigated the effect of 2 factors (laying dateand parental condition) that could have mediated the shift inthe effect of social dominance on sex allocation in the courseof the study. Laying date was positively associated with theproportion of males, but this effect was independent of socialdominance. Maternal condition (residual mass over tarsus andegg volume) was related to social dominance but not to clutchsex ratio. Paternal condition (residual mass over tarsus) wasnot related to clutch sex ratio. We discuss how spatial or temporalvariation in effects of variables such as social dominance onsex allocation can contribute to our understanding of the evolutionof sex allocation in species with complex life histories.     

Genetic and environmental influences on self-reported and parent-reported behavior problems in young adult adoptees

The aim of the present study was to estimate the genetic, shared and nonshared environmental contributions to self-reported and parent-reported internalizing and externalizing problems in a follow-up study of intercountry adopted young adults. Young Adult Self-Report ratings were obtained from 1475 adoptees aged 22–32 years and Young Adult Behavior Checklist ratings from 1115 adoptive parents. For the genetic analyses, a subset of 143 adopted biologically related and 295 unrelated siblings was used. The data were subjected to model fitting decomposing three sources of variance: genetic factors (A) shared environment (C) and nonshared environment (E). Genetic factors were of more importance in both self-reported ( A ²= 54%, C ²= 0, and E ²= 46%) and parent-reported ( A ²= 76%, C ²= 15% and E ²= 9%) internalizing problems. Environmental factors were of more importance in both self-reported ( A ²= 33%, C ²= 17% and E ²= 50%) and parent-reported ( A ²= 28%, C ²= 27% and E ²= 45%) externalizing problems. This was in contrast with findings from the first and second assessments in the same sample during adolescence when genetic factors were more important in explaining externalizing problems compared with internalizing problems. Our results suggest a developmental reversal in genetic and environmental influences on behavior problems from early adolescence into adulthood, which could be related to different underlying developmental trajectories.     

Mucosal response in African catfish after administration of Vibrio anguillarum O2 antigens via different routes

The aim of this study was to investigate the possibility of mucosal vaccination in African catfish (Clarias gariepinus) with Vibrio anguillarum O2 bacterins. The antigen was administered via different routes: anal intubation, oral administration, intraperitoneal injection and immersion. To monitor the antigen uptake, a competitive ELISA was used. The antibody response was measured using an indirect ELISA. Increased antibody levels were found in bile and mucus upon anal intubation, which was not the case after intraperitoneal injection. The data indicate that oral vaccination of fish may be possible when antigens can reach the second gut segment in sufficient quantities and in the right form as confirmed by the recorded substantial induction of systemic and mucosal immunity. The results obtained are a strong indication for mucosal immune response and the two compartmental models for immune response in fish.     

In vitro analysis of interferon gamma (IFN-gamma) and interleukin-12 (IL-12) production and their effects in ileal Crohn's disease

Crohn's disease is an inflammatory disease of the gut in which tumour necrosis factor (TNF) and T helper 1 (Th1) cytokines (interleukin (IL)-12, interferon (IFN)-gamma) are thought to play a major role. After the successes obtained with neutralisation of TNF, interest is now growing for therapy aiming at neutralisation of Th1-associated cytokines. Since cytokines are linked in a delicate network, in vitro cultures of ileal lamina propria mononuclear cells (LPMC) were set up for evaluation of a) IFN-gamma and IL-12 production, b) effects of rhIFN-gamma and rhIL-12 and c) effects of anti-IFN-gamma and anti-IL-12 on pro-inflammatory cytokines and IL-10 production. LPMC were isolated from surgical specimens of a total of 27 Crohn's disease and 17 caecum carcinoma (control) patients. Cells were stimulated with CD40L (which triggers myeloid CD40-expressing cells) or anti-CD3 +CD80 (which triggers T cells). LPMC from involved ileal, Crohn's disease produced, in both non-stimulated and stimulated conditions, more IFN-gamma and IL-12p70 than LPMC from non-involved tissue or from control patients. rhIFN-gamma significantly enhanced TNF production in both controls and in ileal Crohn's disease patients, while rhIL-12 enhanced IFN-gamma but not TNF production. LPMC from involved tissue were more sensitive to IL-12 than control LPMC. LP-T cell-dependent activation of monocytes was then studied by co-culture of anti-CD3/CD80-stimulated LPMC with fresh monocytes, which resulted in high IL-12, IFN-gamma, TNF and IL-10 production. The data show that neutralisation of either IL-12 or IFN-gamma with mAb in these cultures also affects secretion of the reciprocal cytokine and (in the case of anti-IL-12) also that of the anti-inflammatory cytokine IL-10. However, no effect of anti-IL-12 or anti-IFN-gamma on production of TNF, a cytokine with an important pathogenic role in Crohn's disease, could be found. Therapies aiming at neutralisation of IFN-gamma or IL-12 are therefore unlikely to replace anti-TNF, but they might provide an additive or synergistic effect.