A modular design for the clathrin- and actin-mediated endocytosis machinery

Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.     

Comparison of two management strategies for Helicobacter pylori treatment: clinical study and cost-effectiveness analysis

BACKGROUND: First-line proton pump inhibitor-based triple and quadruple therapies for Helicobacter pylori eradication present similar levels of efficacy. Cross-over treatment (quadruple following triple failure, and triple following quadruple failure) seems the most sensible approach to treatment failures, but the two strategies -'quadruple first' versus 'triple first'- have not been previously compared. The aims of our study were to assess the usefulness and the cost-effectiveness of the two treatment strategies. MATERIAL AND METHODS: Forty-nine out of 344 patients included in a previous study comparing triple therapy - 7 days of omeprazole, amoxicillin and clarithromycin twice a day - with quadruple therapy - 7 days of omeprazole twice a day, plus tetracycline, metronidazole and bismuth subcitrate three times a day - failed initial treatment and were assigned to cross-over therapy. Cure was determined by urea breath test. A decision analysis was performed to compare the two eradication strategies. RESULTS: Intention to treat cure rates were 46% (10/22 patients; 95% CI 24-68%) for second-line triple therapy and 63% (17/27 patients; 95% CI 42-81%) for second-line quadruple therapy. Per protocol cure rates were 71% and 85%, respectively. Intention to treat cure rates were 87% (95% CI 81-92%) for the 'triple first' versus 86% (95% CI 80-91%) for the 'quadruple first' strategy (p = .87). The 'quadruple first' strategy was more cost-effective. The incremental cost of 'triple first' strategy per person was 19 in the low-cost area and 65 US dollars in the high-cost area. CONCLUSIONS: The effectiveness of 'triple first' and 'quadruple first' strategies is similar, although the latter seems slightly more cost-effective.     

In silico fragment-based discovery of indolin-2-one analogues as potent DNA gyrase inhibitors

We describe here the fragment-based design of potent DNA gyrase inhibitors. Using the tools of virtual screening and NMR spectroscopy we identified the binding of two low-molecular weight fragments (2-aminobenzimidazole and indolin-2-one) to the 24kDa N-terminal fragment of DNA gyrase B. Further in silico optimization of indolin-2-one led to the discovery of potent DNA gyrase inhibitors.     

Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.     

Clash of kingdoms or why Drosophila larvae positively respond to fungal competitors

BACKGROUND: Competition with filamentous fungi has been demonstrated to be an important cause of mortality for the vast group of insects that depend on ephemeral resources (e.g. fruit, dung, carrion). Recent data suggest that the well-known aggregation of Drosophila larvae across decaying fruit yields a competitive advantage over mould, by which the larvae achieve a higher survival probability in larger groups compared with smaller ones. Feeding and locomotor behaviour of larger larval groups is assumed to cause disruption of fungal hyphae, leading to suppression of fungal growth, which in turn improves the chances of larval survival to the adult stage. Given the relationship between larval density, mould suppression and larval survival, the present study has tested whether fungal-infected food patches elicit communal foraging behaviour on mould-infected sites by which larvae might hamper mould growth more efficiently. RESULTS: Based on laboratory experiments in which Drosophila larvae were offered the choice between fungal-infected and uninfected food patches, larvae significantly aggregated on patches containing young fungal colonies. Grouping behaviour was also visible when larvae were offered only fungal-infected or only uninfected patches; however, larval aggregation was less strong under these conditions than in a heterogeneous environment (infected and uninfected patches). CONCLUSION: Because filamentous fungi can be deadly competitors for insect larvae on ephemeral resources, social attraction of Drosophila larvae to fungal-infected sites leading to suppression of mould growth may reflect an adaptive behavioural response that increases insect larval fitness and can thus be discussed as an anti-competitor behaviour. These observations support the hypothesis that adverse environmental conditions operate in favour of social behaviour. In a search for the underlying mechanisms of communal behaviour in Drosophila, this study highlights the necessity of investigating the role of inter-kingdom competition as a potential driving force in the evolution of spatial behaviour in insects.     

Vesicle mobility studied in cultured astrocytes

Astrocytes release many neuroactive substances, which are stored in membrane bound vesicles and may play a role in synapse modulation and in the coupling between neuronal activity and the local blood flow. However, the mobility of these vesicles in astrocytes has not been studied yet. We here used a fluorescently tagged proatrial natriuretic peptide to label single vesicles and dynamic microscopy to monitor their mobility. To track and analyze labeled vesicles, we employed a computer software. We found two modes of vesicle mobility, directional and non-directional. The mobility of non-directional vesicles is likely determined mainly by free diffusion. Only directional vesicles displayed a straight-line motion. The relationship of mean square displacement with time in directional vesicles resembled a quadratic function, indicating that in addition to free diffusion other mechanisms may contribute to vesicle movements in astrocytes, the biophysical properties of which are similar to those of neurons.     

MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages

Alveolar macrophages (AMs) avidly bind and ingest inhaled environmental particles and bacteria. To identify the particle binding receptor(s) on human AMs, we used functional screening of anti-human AM hybridomas and isolated a mAb, PLK-1, which inhibits AM binding of unopsonized particles (e.g., TiO2, latex beads; 63 +/- 5 and 67 +/- 4% inhibition, respectively, measured by flow cytometry; n = 11) and unopsonized bacteria ( approximately 84 and 41% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PLK-1, respectively). The PLK-1 Ag was identified as the human class A scavenger receptor (SR) MARCO (macrophage receptor with collagenous structure) by observing specific immunolabeling of COS cells transfected with human MARCO (but not SR-AI/II) cDNA and by immunoprecipitation by PLK-1 of a protein of appropriate molecular mass (approximately 70 kDa) from both normal human bronchoalveolar lavage cells (>90% AMs) and human MARCO-transfected COS cells. PLK-1 also specifically inhibited particle binding by COS cells, only after transfection with human MARCO cDNA. Immunostaining showed specific labeling of AMs within human lung tissue, bronchoalveolar lavage samples, as well as macrophages in other sites (e.g., lymph node and liver). Using COS transfectants with different truncated forms of MARCO, allowed epitope mapping for the PLK-1 Ab to MARCO domain V between amino acid residues 420 and 431. A panel of Abs to various SRs identified expression on AMs, but failed to inhibit TiO2 or S. aureus binding. The data support a dominant role for MARCO in the human AM defense against inhaled particles and pathogens.     

Micromechanical analysis of the binding of DNA-bending proteins HMGB1, NHP6A, and HU reveals their ability to form highly stable DNA-protein complexes

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate. In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force. Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding. HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs. Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds. Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact. We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain.     

Lucigenin and coelenterazine as superoxide probes in mitochondrial and bacterial membranes

The chemiluminescent superoxide indicators lucigenin and coelenterazine were compared in rat liver submitochondrial particles and cytoplasmic membranes from Paracoccus denitrificans. Qualitative monitoring is possible with both probes, but quantitative work with lucigenin is hampered by its dependence on one-electron reduction before the photon-emitting reaction. Therefore, calibration of measurements on complex I, capable of efficient lucigenin prereduction with reduced nicotinamide adenine dinucleotide, against xanthine oxidase, which in the presence of hypoxanthine is not able to reduce the probe to a significant rate compared to complex I, may give results in error by one order of magnitude. Coelenterazine, although susceptible of storage-dependent high background chemiluminescence, does not require prereduction and is thus a more reliable probe.