Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of alphaVbeta3-expressing endothelial cells

The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.     

How translation termination factor eRF1 Euplotes does not recognise UGA stop codon

In universal-code eukaryotes, a single class-1 translation termination factor eRF1 decodes all three stop codons, UAA, UAG, and UGA. In some ciliates with variant genetic codes one or two stop codons are used to encode amino acid(s) and are not recognized by eRF1. In Stylonychia, UAG and UAA codons are reassigned as glutamine codons, and in Euplotes, UGA is reassigned as cysteine codon. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of eRF1. Because variant-code ciliates most likely evolved from universal code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. To find out amino acid residues which confer UAR-only specificity to Euplotes aediculatus eRF1, eRFI chimeras were constructed by swapping eRF1 E. aediculatus N-terminal domain sequences with the matching ones from the human protein. In these chimeras the MC-domain was from human eRF1. Functional analysis of these chimeric eRFI highlighted the crucial role of the two regions (positions 38-50 and 123-145) in the N-terminal domain of E. aediculatus eRF1 that restrict E. aediculatus eRF1 specificity toward UAR codons. Possibly, restriction of eRF1 specificity to UAR codons might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UGA to sense codons.     

Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules

Mitochondrion movement and positioning was studied in elongating cultured cells of tobacco (Nicotiana tabacum L.), containing mitochondria-localized green fluorescent protein. In these cells mitochondria are either actively moving in strands of cytoplasm transversing or bordering the vacuole, or immobile positioned in the cortical layer of cytoplasm. Depletion of the cell's ATP stock with the uncoupling agent DNP shows that the movement is much more energy demanding than the positioning. The active movement is F-actin based. It is inhibited by the actin filament disrupting drug latrunculin B, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime and the sulphydryl-modifying agent N-ethylmaleimide. The microtubule disrupting drug oryzalin did not affect the movement of mitochondria itself, but it slightly stimulated the recruitment of cytoplasmic strands, along which mitochondria travel. The immobile mitochondria are often positioned along parallel lines, transverse or oblique to the cell axis, in the cortical cytoplasm of elongated cells. This positioning is mainly microtubule based. After complete disruption of the F-actin, the mitochondria parked themselves into conspicuous parallel arrays transverse or oblique to the cell axis or clustered around chloroplasts and around patches and strands of endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel arrays.     

The mdoC Gene of Escherichia coli Encodes a Membrane Protein That Is Required for Succinylation of Osmoregulated Periplasmic Glucans

Osmoregulated periplasmic glucans (OPGs) of Escherichia coli are anionic oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by phosphoglycerol originating from the membrane phospholipids and by succinyl residues from unknown origin. A phosphoglycerol-transferase-deficient mdoB mutant was subjected to Tn5 transposon mutagenesis, and putative mutant clones were screened for changes in the anionic character of OPGs by thin-layer chromatography. One mutant deficient in succinylation of OPGs was obtained, and the gene inactivated in this mutant was characterized and named mdoC. mdoC, which encodes a membrane-bound protein, is closely linked to the mdoGH operon necessary for the synthesis of the OPG backbone.     

Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.     

C57BL/6J and DBA/2J mice vary in lick rate and ingestive microstructure

Fluid licking in mice is an example of a rhythmic behavior thought to be under the control of a central pattern generator. Inbred strains of mice have been shown to differ in mean or modal interlick interval (ILI) duration, suggesting a genetic-based variation. We investigated water licking in the commonly used inbred strains C57BL/6J (B6) and DBA/2J (D2), using a commercially available contact lickometer. Results from 20-min test sessions indicated that D2 mice lick at a faster rate than B6 mice (10.6 licks/s vs. 8.5 licks/s), based on analysis of the distribution of short-duration ILIs (50-160 ms). This strain difference was independent of sex, extent of water deprivation or total number of licks. D2 mice also displayed a faster lick rate when the strains were tested with a series of brief (5 s) trials. However, when ingestion over the entire 20-min session was analyzed, it was evident that D2 mice had an overall slower rate of ingestion than B6 mice. This was because of the tendency for D2 mice to have more very long pauses (>30 s) between sequences of licking bursts. Overall, it appeared that D2 mice licked more efficiently, ingesting more rapidly during excursions to the spout that were fewer and farther between.     

Enzyme-catalyzed gel proteolysis: an anomalous diffusion-controlled mechanism

Enzyme-catalyzed proteolysis of gelatin gels has been studied. We report a gel degradation rate varying as the square of the enzyme concentration. The diffusion motion of enzymes in the gel has been measured by two-photon fluorescence correlation spectroscopy and identified as being anomalously slow. These experimental results are discussed from a theoretical point of view and interpreted in terms of a diffusion-controlled mechanism for the gel degradation. These results make a step toward the understanding of enzyme-catalyzed gel degradation and give new insight on biological processes such as the action of metalloproteinases in the extracellular matrix involved in cellular invasion.