Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.     

Changes in the auditory neuropil after deafferentation in adult grasshoppers (Schistocerca gregaria)

Nervous systems are capable of structural adjustments. Such plastic changes also occur in the auditory system of the locust Schistocerca gregaria in which a deafferentation leads to compensatory mechanisms, such as collateral sprouting of interneurons. In this study we further investigated lesion related changes in the major auditory neuropil, the median ventral association center (mVAC) of the metathoracic ganglion. The auditory sensory organ of adult locusts was unilaterally extirpated and the mVAC was histologically and immunocytochemically analyzed until 20 days postoperative. Measurements of the neuropil area in transverse sections showed a decrease in size. The putative transmitter of the afferents, acetylcholine, was investigated by acetylcholinesterase histochemistry. Comparisons of staining intensities in the intact and deafferentated mVAC indicated that the amount of acetylcholinesterase in the deafferentated mVAC decreased shortly after the operation. Both, the decreases in size of the mVAC as well as that in acetylcholinesterase histochemistry were only less than 10% compared to the controls. The immunoreactivity against the neurotransmitters γ-amino butyric acid and serotonin was not influenced by the deafferentation.     

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.     

Missorting of LaCrosse virus nucleocapsid protein by the interferon-induced MxA GTPase involves smooth ER membranes

The interferon-induced human MxA protein belongs to the class of dynamin-like, large guanosine-5'-triphosphatases that are involved in intracellular vesicle trafficking and organelle homeostasis. MxA shares many properties with the other members of this protein superfamily, including the propensity to self-assemble and to associate with lipid membranes. However, MxA is unique in that it has antiviral activity and inhibits the replication of several RNA viruses. Here, we determined the role of membranes for the antiviral function of MxA using LaCrosse-bunyavirus (LACV). We show that MxA does not affect trafficking and sorting of viral glycoproteins but binds and mislocates the viral nucleocapsid (N) protein into membrane-associated, large perinuclear complexes. We further demonstrate that MxA localizes to a subcompartment of the smooth endoplasmic reticulum where the viral N protein accumulates. In infected MxA-expressing cells, oligomeric MxA/N complexes are formed in close association with COP-I-positive vesicular-tubular membranes. Our results suggest that this membrane compartment is the preferred place where MxA and N interact, leading to efficient sequestration and missorting of an essential viral component.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

d-matrix – database exploration,visualization and analysis

Background  

Motivated by a biomedical database set up by our group, we aimed to develop a generic database front-end with embedded knowledge discovery and analysis features. A major focus was the human-oriented representation of the data and the enabling of a closed circle of data query, exploration, visualization and analysis.     

Regulation of sister chromatid cohesion between chromosome arms

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.