A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics,Spindle Orientation and Proper Chromosome Segregation during Mitosis

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.     

Source Memory for Mental Imagery: Influences of the Stimuli’s Ease of Imagery

The present study investigated how ease of imagery influences source monitoring accuracy. Two experiments were conducted in order to examine how ease of imagery influences the probability of source confusions of perceived and imagined completions of natural symmetric shapes. The stimuli consisted of binary pictures of natural objects, namely symmetric pictures of birds, butterflies, insects, and leaves. The ease of imagery (indicating the similarity of the sources) and the discriminability (indicating the similarity of the items) of each stimulus were estimated in a pretest and included as predictors of the memory performance for these stimuli. It was found that confusion of the sources becomes more likely when the imagery process was relatively easy. However, if the different processes of source monitoring—item memory, source memory and guessing biases—are disentangled, both experiments support the assumption that the effect of decreased source memory for easily imagined stimuli is due to decision processes and misinformation at retrieval rather than encoding processes and memory retention. The data were modeled with a Bayesian hierarchical implementation of the one high threshold source monitoring model.     

Characterization of endocytic compartments after holo-high density lipoprotein particle uptake in HepG2 cells

Holo-high density lipoprotein (HDL) particle uptake, besides selective lipid uptake, constitutes an alternative pathway to regulate cellular cholesterol homeostasis. In the current study, the cellular path of holo-HDL particles was investigated in human liver carcinoma cells (HepG2) using combined light and electron microscopical methods. The apolipoprotein moiety of HDL was visualized with different markers: horseradish peroxidase, colloidal gold and the fluorochrome Alexa⁵⁶⁸, used in fluorescence microscopy and after photooxidation correlatively at the ultrastructural level. Time course experiments showed a rapid uptake of holo-HDL particles, an accumulation in endosomal compartments, with a plateau after 1–2 h of continuous uptake, and a clearance 1–2 h upon replacement by unlabeled HDL. Correlative microscopy, using HDL-Alexa⁵⁶⁸-driven diaminobenzidine (DAB) photooxidation, identified the fluorescent organelles as DAB-positive multivesicular bodies (MVBs) in the electron microscope; their luminal contents but not the internal vesicles were stained. Labeled MVBs increased in numbers and changed shapes along with the duration of uptake, from polymorphic organelles with multiple surface domains and differently shaped protrusions dominating at early times of uptake to compact bodies with mainly tubular appendices and densely packed vesicles after later times. Differently shaped and labeled surface domains and appendices, as revealed by three dimensional reconstructions, as well as images of homotypic fusions indicate the dynamics of the HDL-positive MVBs. Double staining visualized by confocal microscopy, along with the electron microscopic data, shows that holo-HDL particles after temporal storage in MVBs are only to a minor degree transported to lysosomes, which suggests that different mechanisms are involved in cellular HDL clearance, including resecretion.     

Long-term stability of marker gene expression in Prunus subhirtella: a model fruit tree species

Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. Since trees are particularly suited for long-term evaluations of the impact of the technology, Prunus subhirtella autumno rosa (PAR) was chosen as model fruit tree species and transformed with a reporter gene (uidA) under the control of the 35S promoter. Using Southern and GUS fluorometric techniques, we compared transgene copy numbers and observed stability of transgene expression levels in 34 different transgenic plants, grown under in vitro, greenhouse and screenhouse conditions, over a period of 9 years. An influence of grafting on gene expression was not observed. No silenced transgenic plant was detected. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs, confirming the value of PAR as model species to study season-dependent regulation in mature stone fruit tissues. While the Agrobacterium-derived Prunus transformants contained one to two copies of the transgenes, 91% of the transgenic events also contained various lengths of the bacterial plasmid backbone, indicating that the Agrobacterium-mediated transformation is not as precise as previously perceived. The implications for public acceptance and future applications are discussed.     

Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab

The major allergens of honeybee venom, hyaluronidase (Hyal) and phospholipase A2, can induce life-threatening IgE-mediated allergic reactions in humans. Although conventional immunotherapy is effective, up to 40% of patients develop allergic side effects including anaphylaxis and thus, there is a need for an improved immunotherapy. A murine monoclonal anti-Hyal IgG1 antibody (mAb 21E11), that competed for Hyal binding with IgEs from sera of bee venom allergic patients, was raised. The fragment of these IgG antibodies which bind to antigen (Fab) was produced and complexed (1:1) with Hyal. The crystal structure determination of Hyal/Fab 21E11 complex (2.6 A) enabled the identification of the Hyal-IgG interface which provides indirect information on the Hyal-IgE interaction (B-cell epitope). The epitope is composed of a linear array of nine residues (Arg138, His141-Arg148) located at the tip of a helix-turn-helix motive which protrudes away from the globular core and fits tightly into the deep surface pocket formed by the residues from the six complementarity determining regions (CDRs) of the Fab. The epitope is continuous and yet its conformation appears to be essential for Ab recognition, since the synthetic 15-mer peptide comprising the entire epitope (Arg138-Glu152) is neither recognized by mAb 21E11 nor by human IgEs. The structure of the complex provides the basis for the rational design of Hyal derivatives with reduced allergenic activity, which could be used in the development of safer allergen-specific immunotherapy.     

Menarcheal onset is associated with body composition parameters but not with socioeconomic status

In the present study the menarcheal status (pre-or postmenarcheal), body composition, weight status, and socioeconomic parameters such as type of school and parental educational level as of 1752 female adolescents ageing between 12 and 18 years (x = 14.6) from rural areas of Eastern Austria were documented. Furthermore the association patterns between body composition, socioeconomic parameters and menarcheal status were analyzed. It turned out, that body composition parameters such as BMI, lean body mass and absolute and relative fat mass were significantly associated with menarcheal status. Postmenarcheal girls were taller and exhibited a higher weight status, a higher absolute and relative amount of fat mass and a higher amount of lean body mass than their premenarcheal counterparts of the same age. In contrast to the significant association between body composition and menarcheal status, no significant impact of educational level on menarcheal status, indicating socioeconomic status could be documented.     

PDE-10A inhibitors as insulin secretagogues

Modulation of cAMP levels has been linked to insulin secretion in preclinical animal models and in humans. The high expression of PDE-10A in pancreatic islets suggested that inhibition of this enzyme may provide the necessary modulation to elicit increased insulin secretion. Using an HTS approach, we have identified quinoline-based PDE-10A inhibitors as insulin secretagogues in vitro. Optimized compounds were evaluated in vivo where improvements in glucose tolerance and increases in insulin secretion were measured.     

Biodegradation of chlorobenzene under hypoxic and mixed hypoxic-denitrifying conditions

Pseudomonas veronii strain UFZ B549, Acidovorax facilis strain UFZ B530, and a community of indigenous groundwater bacteria, adapted to oxygen limitation, were cultivated on chlorobenzene and its metabolites 2-chloro-cis,cis-muconate and acetate/succinate under hypoxic and denitrifying conditions. Highly sensitive approaches were used to maintain defined low oxygen partial pressures in an oxygen-re-supplying headspace. With low amounts of oxygen available all cultures converted chlorobenzene, though the pure strains accumulated 3-chlorocatechol and 2-chloro-cis,cis-muconate as intermediates. Under strictly anoxic conditions no chlorobenzene transformation was observed, while 2-chloro-cis,cis-muconate, the fission product of oxidative ring cleavage, was readily degraded by the investigated chlorobenzene-degrading cultures at the expense of nitrate as terminal electron acceptor. Hence, we conclude that oxygen is an obligatory reactant for initial activation of chlorobenzene and fission of the aromatic ring, but it can be partially replaced by nitrate in respiration. The tendency to denitrify in the presence of oxygen during growth on chlorobenzene appeared to depend on the oxygen availability and the efficiency to metabolize chlorobenzene under oxygen limitation, which is largely regulated by the activity of the intradiol ring fission dioxygenase. Permanent cultivation of a groundwater consortium under reduced oxygen levels resulted in enrichment of a community almost exclusively composed of members of the β-Proteobacteria and Bacteroidetes. Thus, it is deduced that these strains can still maintain high activities of oxygen-requiring enzymes that allow for efficient CB transformation under hypoxic conditions.     

Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae

RAB5 GTPases are important regulators of endosomal membrane traffic. Among them Arabidopsis thaliana ARA7/RABF2b is highly conserved and homologues are present in fungal, animal and plant kingdoms. In land plants ARA7 and its homologues are involved in endocytosis and transport towards the vacuole. Here we report on the isolation of an ARA7 homologue (CaARA7/CaRABF2) in the highly evolved characean green alga Chara australis. It encodes a polypeptide of 202 amino acids with a calculated molecular mass of 22.2 kDa and intrinsic GTPase activity. Immunolabelling of internodal cells with a specific antibody reveals CaARA7 epitopes at multivesicular endosomes (MVEs) and at MVE‐containing wortmannin (WM) compartments. When transiently expressed in epidermal cells of Nicotiana benthamiana leaves, fluorescently tagged CaARA7 localizes to small organelles (putative MVEs) and WM compartments, and partially colocalizes with AtARA7 and CaARA6, a plant specific RABF1 GTPase. Mutations in membrane anchoring and GTP binding sites alter localization of CaARA7 and affect GTPase activity, respectively. This first detailed study of a conventional RAB5 GTPase in green algae demonstrates that CaARA7 is similar to RAB5 GTPases from land plants and other organisms and shows conserved structure and localization.