TRAIL-induced apoptosis is independent of the mitochondrial apoptosis mediator DAP3

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and the adapter protein Fas-associated death domain protein (FADD). Recently, an adapter function for death-associated protein 3 (DAP3) between DR4/DR5 and FADD has been proposed. However, DAP3 has been reported to be a ribosomal protein localized to the mitochondrial matrix. To address these discrepancies, the intracellular localization of DAP3 after apoptosis induction in human T-lymphocytes with recombinant TRAIL was analyzed. DAP3, in contrast to cytochrome c, remained intra-mitochondrial during apoptosis. No interaction between FADD and DAP3 after cell fractionation could be detected as long as subcellular compartments remained intact. Only whole cell lysate co-immunoprecipitation revealed an ex vivo interaction between DAP3 and FADD. Therefore, DAP3 and FADD interact only in vitro after disruption of the cellular compartments. TRAIL-induced and DR4-mediated apoptosis in Jurkat cells is independent of DAP3.     

Unstable kinetochore-microtubule capture and chromosomal instability following deletion of CENP-E

A selective disruption of the mouse CENP-E gene was generated to test how this kinetochore-associated, kinesin-like protein contributes to chromosome segregation. The removal of CENP-E in primary cells produced spindles in which some metaphase chromosomes lay juxtaposed to a spindle pole, despite the absence of microtubules stably bound to their kinetochores. Most CENP-E-free chromosomes moved to the spindle equator, but their kinetochores bound only half the normal number of microtubules. Deletion of CENP-E in embryos led to early developmental arrest. Selective deletion of CENP-E in liver revealed that tissue regeneration after chemical damage was accompanied by aberrant mitoses marked by chromosome missegregation. CENP-E is thus essential for the maintenance of chromosomal stability through efficient stabilization of microtubule capture at kinetochores.     

90S pre-ribosomes include the 35S pre-rRNA,the U3 snoRNP,and 40S subunit processing factors but predominantly lack 60S synthesis factors

We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.     

Psychosocial adaptation to short stature - an indication for growth hormone therapy?

Although growth hormone does not clearly improve final height in non-growth-hormone-deficient children with short stature, it leads to a temporary acceleration of growth velocity. It is an ongoing discussion whether this effect supports psychosocial adaptation to short stature and therefore could be an indication for growth hormone treatment in children with short stature without growth hormone deficiency. We have reviewed recent literature concerning psychosocial consequences of short stature. Together with own data we can demonstrate that short people regularly adapt well to their height and have a good self-esteem. On the other hand, we focus on the problem that most studies on this subject suffer from methodical problems. A growth-related questionnaire that evaluates subjective and objective perceptions of being short in patients and peers is not at hand. As a consequence, psychosocial problems due to short stature have not been exactly classified yet and therefore do not represent an indication for growth hormone therapy.     

Isoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display library

Epitopes differing among isoenzymes of creatine kinase (CK) are apparently limited in number and poorly immunogenic in vivo. Especially for the BB-CK isoenzyme, very few monoclonal antibodies (mAb) are available. Here, we use in vitro selection with a synthetic human phage display antibody library and develop isoenzyme competition and peptide panning strategies to obtain human single chain Fv (scFv) antibodies against specific CK isoenzymes. We isolated and characterized seven scFv clones that recognize native as well as denatured cytosolic BB-CK in ELISA, immunoblot, immunofluorescence histochemistry and surface plasmon resonance (SPR) spectroscopy. To a variable but minor degree, they also react with cytosolic MM-CK, but not with mitochondrial CK isoenzymes. Epitope mapping revealed that the scFv antibodies recognize different BB-CK epitopes, including the N-terminus and the isoenzyme-specific box, a highly conserved sequence of unknown function for which no mAb were available so far. With a K(D) of 3.5-9.6 x 10(-7) M, the isolated scFv compare favorably with mouse mAb and may overcome certain of their limitations. Our results demonstrate the advantages of in vitro antibody selection for the generation of isoenzyme-specific antibodies.     

Globin genes are present in Ciona intestinalis

The key position of the Ciona intestinalis basal to the vertebrate phylogenetic tree brings up the question of which respiratory proteins are used by the tunicate to facilitate oxygen transport and storage. The publication of the Ciona draft genome sequence suggests that globin genes are completely missing and that-like some molluscs and arthropods-the sea squirt uses hemocyanin instead of hemoglobin for respiration. However, we report here the presence and expression of at least four distinct globin gene/protein sequences in Ciona. This finding is in agreement with the ancestral phylogeny of the vertebrate globins. Moreover, it seems likely that the Ciona hemocyanin-like sequences have enzymatic instead of respiratory functions.     

Survival and growth of Francisella tularensis in Acanthamoeba castellanii

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.