Regulation of translation is required for dendritic cell function and survival during activation

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. Here, we show that in response to lipopolysaccharides, protein synthesis is rapidly enhanced in DCs. This enhancement occurs via a PI3K-dependent signaling pathway and is key for DC activation. In addition, we show that later on, in a manner similar to viral or apoptotic stress, DC activation leads to the phosphorylation and proteolysis of important translation initiation factors, thus inhibiting cap-dependent translation. This inhibition correlates with major changes in the origin of the peptides presented by MHC class I and the ability of mature DCs to prevent cell death. Our observations have important implications in linking translation regulation with DC function and survival during the immune response.     

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.     

Regulation of Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii

The anaerobic acetogenic bacterium Acetobacterium woodii can conserve energy by oxidation of various substrates coupled to either carbonate or caffeate respiration. We used a cell suspension system to study the regulation and kinetics of induction of caffeate respiration. After addition of caffeate to suspensions of fructose-grown cells, there was a lag phase of about 90 min before caffeate reduction commenced. However, in the presence of tetracycline caffeate was not reduced, indicating that de novo protein synthesis is required for the ability to respire caffeate. Induction also took place in the presence of CO₂, and once a culture was induced, caffeate and CO₂ were used simultaneously as electron acceptors. Induction of caffeate reduction was also observed with H₂ plus CO₂ as the substrate, but the lag phase was much longer. Again, caffeate and CO₂ were used simultaneously as electron acceptors. In contrast, during oxidation of methyl groups derived from methanol or betaine, acetogenesis was the preferred energy-conserving pathway, and caffeate reduction started only after acetogenesis was completed. The differential flow of reductants was also observed with suspensions of resting cells in which caffeate reduction was induced prior to harvest of the cells. These cell suspensions utilized caffeate and CO₂ simultaneously with fructose or hydrogen as electron donors, but CO₂ was preferred over caffeate during methyl group oxidation. Caffeate-induced resting cells could reduce caffeate and also p-coumarate or ferulate with hydrogen as the electron donor. p-Coumarate or ferulate also served as an inducer for caffeate reduction. Interestingly, caffeate-induced cells reduced ferulate in the absence of an external reductant, indicating that caffeate also induces the enzymes required for oxidation of the methyl group of ferulate.     

Recycling endosomes in neuronal membrane traffic

Neurons are highly polarized cells with axonal and somatodendritic membrane surfaces that spatially separate signal-sending from signal-receiving membrane domains. As found in many other cell types, different populations of endosomes are involved in the sorting of synaptic and other membrane cargo in neurons. The exact source of the membrane for neurite extension and process remodelling during neuronal differentiation has remained uncertain, and we do not know exactly how polarized sorting of neuronal membrane proteins is achieved. In the present article, we will provide a brief overview of endosomes and their putative or proven functions in fibroblasts, epithelial cells and neurons. On the basis of insights from non-neuronal cell types and recent studies on the function of recycling endosomes during synaptic plasticity-induced membrane remodelling, we postulate a speculative model regarding the role of recycling endosomes in neuronal differentiation.     

Innervation pattern of suboesophageal ventral unpaired median neurones in the honeybee brain

In honeybees (Apis mellifera), the biogenic amine octopamine has been shown to play a role in associative and non-associative learning and in the division of labour in the hive. Immunohistochemical studies indicate that the ventral unpaired median (VUM) neurones in the suboesophageal ganglion (SOG) are putatively octopaminergic and therefore might be involved in the octopaminergic modulation of behaviour. In contrast to our knowledge about the behavioural effects of octopamine, only one neurone (VUMmx1) has been related to a behavioural effect (the reward function during olfactory learning). In this study, we have investigated suboesophageal VUM neurones with fluorescent dye-tracing techniques and intracellular recordings combined with intracellular staining. Ten different VUM neurones have been found including six VUM neurones innervating neuropile regions of the brain and the SOG exclusively (central VUM neurones) and four VUM neurones with axons in peripheral nerves (peripheral VUM neurones). The central VUM neurones innervate the antennal lobes, the protocerebral lobes (including the lateral horn) and the mushroom body calyces. Of these, a novel mandibular VUM neurone, VUMmd1, exhibits the same branching pattern in the brain as VUMmx1 and responds to sucrose and odours in a similar way. The peripheral VUM neurones innervate the antennal and the mandibular nerves. In addition, we describe one labial unpaired median neurone with a dorsal cell body, DUMlb1. The possible homology between the honeybee VUM neurones and the unpaired median neurones in other insects is discussed. This work was supported by the DFG ME 365/24-2.     

Phytoconstituents from the root of Streptocaulon tomentosum and their chemotaxonomical relevance for separation from S. juventas

A new cardenolide, 17β-H-periplogenin-3-O-β-d-digitoxoside (1), and a new pregnane glycoside, Δ⁵-pregnene-3β,16α-diol-d-O-[2,4-O-diacetyl-β-digitalopyranosyl-(1 → 4)-β-d-cymaropyranoside]-16-O-[β-d-glucopyranoside] (2) were isolated from the roots of Streptocaulon tomentosum (Asclepiadaceae) together with a series of known compounds. Their chemotaxonomic significance for the separation of S. tomentosum from Streptocaulon juventas is discussed, suggesting a rather clear distinction of these species.     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Cell death in leukemia: passenger protein regulation by topoisomerase inhibitors

Etoposide is a potent inducer of mitotic catastrophe; a type of cell death resulting from aberrant mitosis. It is important in p53 negative cells where p53 dependent apoptosis and events at the G1 and G2 cell cycle checkpoints are compromised. Passenger proteins regulate many aspects of mitosis and siRNA interference or direct inhibition of Aurora B kinase results in mitotic catastrophe. However, there is little available data of clinical relevance in leukaemia models. Here, in p53 negative K562 myeloid leukemia cells, etoposide-induced mitotic catastrophe is shown to be time and/or concentration dependent. Survivin and Aurora remained bound to chromosomes. Survivin and Aurora were also associated with Cdk1 and were shown to form complexes, which in pull down experiments, included INCENP. There was no evidence of Aurora B kinase suppression. These data suggests etoposide will complement Aurora B kinase inhibitors currently in clinical trials for cancer.     

The role of tryptophan residues in the function and stability of the mechanosensitive channel MscS from Escherichia coli

Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with phenylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability.