Emotion recognition in children and adolescents with attention-deficit/hyperactivity disorder (ADHD)

Children with attention-deficit/hyperactivity disorder (ADHD) are impaired in social adaptation and display deficits in social competence. Deficient emotion recognition has been discussed to underlie these social problems. However, comorbid conduct problems have not been considered in the majority of studies conducted so far, and the influence of medication on emotion recognition has rarely been studied. Here, emotion recognition performance was assessed in children with ADHD without medication compared with children with ADHD under stimulant medication and a matched control group. In order to rule out confounding by externalizing symptoms, children with comorbid conduct problems were excluded. Video clips with neutral faces developing a basic emotion (happiness, sadness, disgust, fear and anger) were presented in order to assess emotion recognition. Results indicated between-group differences neither concerning the number of correctly identified emotions nor concerning reaction times and their standard deviations. Thus, we suggest that ADHD per se is not associated with deficits in emotion recognition.     

BCL-2 is dispensable for thrombopoiesis and platelet survival

Navitoclax (ABT-263), an inhibitor of the pro-survival BCL-2 family proteins BCL-2, BCL-X_L and BCL-W, has shown clinical efficacy in certain BCL-2-dependent haematological cancers, but causes dose-limiting thrombocytopaenia. The latter effect is caused by Navitoclax directly inducing the apoptotic death of platelets, which are dependent on BCL-X_L for survival. Recently, ABT-199, a selective BCL-2 antagonist, was developed. It has shown promising anti-leukaemia activity in patients whilst sparing platelets, suggesting that the megakaryocyte lineage does not require BCL-2. In order to elucidate the role of BCL-2 in megakaryocyte and platelet survival, we generated mice with a lineage-specific deletion of Bcl2, alone or in combination with loss of Mcl1 or Bclx. Platelet production and platelet survival were analysed. Additionally, we made use of BH3 mimetics that selectively inhibit BCL-2 or BCL-X_L. We show that the deletion of BCL-2, on its own or in concert with MCL-1, does not affect platelet production or platelet lifespan. Thrombocytopaenia in Bclx-deficient mice was not affected by additional genetic loss or pharmacological inhibition of BCL-2. Thus, BCL-2 is dispensable for thrombopoiesis and platelet survival in mice.Platelets are anucleate blood cells that play essential roles in haemostasis, wound healing and a range of other processes, including inflammation and immunity.¹ They are produced by megakaryocytes, large polyploid cells that develop primarily in the bone marrow, spleen and foetal liver.² Recent work has demonstrated that the survival of megakaryocytes and platelets is governed by the BCL-2 family proteins.³ Both cell types possess a classical BAK/BAX-mediated intrinsic apoptosis pathway that must be restrained in order for them to develop and survive.In platelets, BCL-X_L is the critical pro-survival BCL-2 family member required to keep BAK and BAX in check. The first evidence of this came from Wagner et al.,⁴ who reported severe thrombocytopaenia in mice after MMTV-Cre-mediated deletion of Bclx in the haematopoietic system, skin and various secretory tissues. It has since been shown that megakaryocyte-restricted deletion of Bclx in mice reduces platelet lifespan from ~5 days to ~5 h, with a concomitant decrease in platelet counts to ~2% of wild-type levels.^{5, 6} Pharmacological inhibition of BCL-X_L with the BH3 mimetics ABT-737⁷ or Navitoclax (ABT-263)⁸ (which both also inhibit BCL-2 and BCL-W) triggers BAK/BAX-mediated platelet apoptosis.^{9, 10, 11} As a result, these drugs cause dose-dependent thrombocytopaenia in mice, dogs and humans.^{9, 11, 12, 13, 14} Indeed, thrombocytopaenia is the dose-limiting toxicity for Navitoclax.^{12, 13, 14} This fact provided additional impetus for the development of agents that specifically target BCL-2, beginning with ABT-199,¹⁵ a BCL-2-selective antagonist currently in clinical trials for the treatment of a range of haematological malignancies including chronic lymphocytic leukaemia, non-Hodgkin''s lymphoma, follicular lymphoma, mantle cell lymphoma, multiple myeloma and acute myeloid leukaemia. ABT-199 has already shown very promising anti-tumour activity, with little to no impact on platelet counts.^{15, 16} These data suggest that BCL-2 is dispensable for the development and survival of platelets.In megakaryocytes, BCL-X_L is also critical for survival. Although not absolutely required for their growth and maturation, BCL-X_L is essential for megakaryocytes to proceed safely through pro-platelet formation and platelet shedding.⁵ In addition to BCL-X_L, megakaryocytes also depend on the pro-survival activity of MCL-1. Conditional deletion of Mcl1 alone has no effect on this lineage. In contrast, combined megakaryocyte-specific loss of Bclx and Mcl1 results in the failure of megakaryopoiesis, systemic haemorrhage and embryonic lethality.^{5, 17, 18} These defects are rescued by deletion of Bak and Bax.¹⁸Consistent with the genetic studies, administration of ABT-737 to Mcl1^Pf4Δ/Pf4Δ mice, which lack MCL-1 in megakaryocytes and platelets, induces acute, fulminant BAK/BAX-dependent megakaryocyte apoptosis. Given that, in addition to BCL-X_L, ABT-737 also targets BCL-2,⁷ these data suggested that BCL-2 might also contribute to the development and survival of the megakaryocyte lineage. This is supported by recent studies demonstrating that neonatal human platelets contain increased levels of BCL-2 relative to adult counterparts,¹⁹ and that platelet lifespan is extended in transgenic mice expressing BCL-2 under the control of the pan-haematopoietic Vav promoter.²⁰ In light of these observations, and intense ongoing activity surrounding the development of novel BH3 mimetics,²¹ we set out to elucidate the role of BCL-2 in megakaryocytes and platelets. Mice with a megakaryocyte-specific deletion of Bcl2, either alone or in combination with deletion of Mcl1 or Bclx, were generated. The effect of these mutations, and of BCL-2 or BCL-X_L-selective BH3 mimetics, on the megakaryocyte lineage was assessed.     

A comparison of root architecture and shoot morphology between naturally regenerated and container-grown seedlings of Quercus ilex

We explored the different mechanisms developed by naturally regenerated seedlings of Quercus ilex L. (Holm oak) under Mediterranean conditions compared to container-seedlings commonly used in plantations. We examined the differences in root architecture (including topology and morphology) and shoot parameters. The results showed that there are many differences in the architecture of the root system as well as in the shoot morphology between the two types of seedlings. The naturally regenerated seedlings were smaller with regard to most of the shoot and root parameters, but they developed a longer taproot, only first order lateral roots, and presented a more herringbone-like root system compared to the container seedlings. Conversely, all types of container seedlings, were larger and had a more extended root system with many orders of lateral roots, while their taproot length was restricted within the container’s depth. The quotient log (α)/ log (μ) for all seedlings, showed a tendency to decrease with plant size. A strict herringbone root system with an elongated taproot may be the optimal root architecture for Quercus ilex L. seedlings in order to survive under Mediterranean conditions.     

Signal peptide peptidase: biochemical properties and modulation by nonsteroidal antiinflammatory drugs

Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves remnant signal peptides after their release by signal peptidase. SPP contains active site motifs also found in presenilin, the catalytic component of the gamma-secretase complex of Alzheimer's disease. However, SPP has a membrane topology opposite that of presenilin, cleaves transmembrane substrates of opposite directionality, and does not require complexation with other proteins. Here we show that, upon isolation of membranes and solubilization with detergent, the biochemical characteristics of SPP are remarkably similar to gamma-secretase. The majority of the SPP-catalyzed cleavages occurred at a single site in a synthetic substrate based on the prolactin (Prl) signal sequence. However, as seen with cleavage of substrates by gamma-secretase, additional cuts at other minor sites are also observed. Like gamma-secretase, SPP is inhibited by helical peptidomimetics and apparently contains a substrate-binding site that is distinct from the active site. Surprisingly, certain nonsteroidal antiinflammatory drugs known to shift the site of proteolysis by gamma-secretase also alter the cleavage site of Prl by SPP. Together, these findings suggest that SPP and presenilin share certain biochemical properties, including a conserved drug-binding site for allosteric modulation of substrate proteolysis.     

Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring--comparison of HPLC with UV-, single MS- and tandem MS-detection

Results of the simultaneous determination of the structurally different antibiotics cefazoline, cefotiame, cefuroxime, chloramphenicol, ciprofloxacin, ofloxacin, sulfamethoxazole and trimethoprim from environmental and biological monitoring using high-performance liquid chromatography with UV, single mass and tandem mass spectrometry were compared. For sample enrichment and clean-up a SPE method using bakerbond C18 cartridges was developed. Mean recovery rates were above 70%. Because of the complex urine matrix, only the wipe samples could be analyzed by UV-detection. However, UV-detection and single MS-detection are useful for control measurements after spillage, e.g. (LOD=1-2 ng/cm(2)). Samples from biological monitoring of occupational uptake should be analyzed by LC-MS/MS. The limits of detection (LOD) in urine ranged from 0.4 to 70 microg/L for LC-MS and 0.01 to 0.9 microg/L for LC-MS/MS detection. The limits of detection in wipe samples ranged from 0.003 to 0.13 ng/cm(2).     

Active gamma-secretase complexes contain only one of each component

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.     

Woodchuck gamma interferon upregulates major histocompatibility complex class I transcription but is unable to deplete woodchuck hepatitis virus replication intermediates and RNAs in persistently infected woodchuck primary hepatocytes.

Gamma interferon (IFN-gamma) is an important mediator with multiple functions in the host defense against viral infection. IFN-gamma, in concert with tumor necrosis factor alpha (TNF-alpha), leads to a remarkable reduction of intrahepatic replication intermediates and specific mRNAs of hepatitis B virus (HBV) by a noncytolytic mechanism in the transgenic mouse model. Thus, it is rational to evaluate the potential value of IFN-gamma for the treatment of chronic HBV infection. In the present study, we expressed recombinant woodchuck IFN-gamma (wIFN-gamma) in Escherichia coli and mammalian cells. wIFN-gamma protected woodchuck cells against infection of murine encephalomyocarditis virus in a species-specific manner. It upregulated the mRNA level of the woodchuck major histocompatibility complex class I (MHC-I) heavy chain in permanent woodchuck WH12/6 cells and regulated differentially the gene expression. However, the level of the replication intermediates and specific RNAs of woodchuck hepatitis virus (WHV) in persistently WHV-infected primary woodchuck hepatocytes did not change despite a treatment with 1,000 U of wIFN-gamma per ml or with a combination of wIFN-gamma and woodchuck TNF-alpha. Rather, hepatocytes derived from chronic carriers had an elevated level of the MHC-I heavy-chain mRNAs, most probably due to the exposure to inflammatory cytokines in vivo. Treatment with high doses of wIFN-gamma led to an abnormal cell morphology and loss of hepatocytes. Thus, wIFN-gamma regulates the gene expression in woodchuck hepatocytes but could not deplete WHV replication intermediates and mRNAs in persistently infected hepatocytes. The cellular response to wIFN-gamma may be changed in hepatocytes from chronically WHV-infected woodchucks. It should be clarified in the future whether the continuous exposure of hepatocytes to inflammatory cytokines or the presence of viral proteins leads to changes of the cellular response to wIFN-gamma.     

Combination of DNA Prime – Adenovirus Boost Immunization with Entecavir Elicits Sustained Control of Chronic Hepatitis B in the Woodchuck Model

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8⁺ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8⁺ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4⁺ and CD8⁺ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients.     

The fossil fallow deer <Emphasis Type="Italic">Dama geiselana</Emphasis> (Cervidae,Mammalia, upgrade to species level) in the context of migration and local extinctions of fallow deer in the Late and Middle Pleistocene in Europe

The fossil fallow deer Dama dama geiselana Pfeiffer from Neumark-Nord (Saxony-Anhalt, Germany) is upgraded to species level and discussed within the current taxonomy of recent and fossil fallow deer. Typical antler and skeletal characteristics are discussed in comparison with the recent Dama dama, Dama mesopotamica and the fossil species Dama clactoniana, Dama nestii and Dama rhenana. The phylogenetic relationship of fallow deer can be traced back to the Late Pliocene and distinguished morphologically from Cervus elaphus, Cervus nippon and Axis axis. Late and Middle Pleistocene finds from Germany are presented and discussed in the context of the finds from the Mediterranean region and Great Britain. The differences in antler morphology and bone dimensions in West German and North-East German fallow deer from the Late Pleistocene support the hypothesis of different immigration channels, on the one hand from the eastern Mediterranean along the Danube and on the other from the west along the Rhone and Rhine. In the Middle Pleistocene, Dama mesopotamica is considered as the typical fallow deer in the eastern Mediterranean, while in the west, Dama clactoniana is widespread. The hypothesis of immigration from the eastern Mediterranean is supported by the fossil record in Germany with the fallow deer from Edesheim. Conversely, Dama geiselana probably influenced East Mediterranean populations. Special tooth characteristics of Dama geiselana occur with lower frequency in Dama mesopotamica. In the Bronze Age, the fallow deer from Kastanas (Macedonia) shares antler characteristics, the high frequency of specific features of the scapula, and the astragalus with Dama geiselana. A relict population of Dama geiselana probably reached the Eastern Mediterranean at the beginning of the last cold stage.