The Perforin Pore Facilitates the Delivery of Cationic Cargos

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.     

Concerted Spatio-Temporal Dynamics of Imported DNA and ComE DNA Uptake Protein during Gonococcal Transformation

Competence for transformation is widespread among bacterial species. In the case of Gram-negative systems, a key step to transformation is the import of DNA across the outer membrane. Although multiple factors are known to affect DNA transport, little is known about the dynamics of DNA import. Here, we characterized the spatio-temporal dynamics of DNA import into the periplasm of Neisseria gonorrhoeae. DNA was imported into the periplasm at random locations around the cell contour. Subsequently, it was recruited at the septum of diplococci at a time scale that increased with DNA length. We found using fluorescent DNA that the periplasm was saturable within minutes with ∼40 kbp DNA. The DNA-binding protein ComE quantitatively governed the carrying capacity of the periplasm in a gene-dosage-dependent fashion. As seen using a fluorescent-tagged derivative protein, ComE was homogeneously distributed in the periplasm in the absence of external DNA. Upon addition of external DNA, ComE was relocalized to form discrete foci colocalized with imported DNA. We conclude that the periplasm can act as a considerable reservoir for imported DNA with ComE governing the amount of DNA stored potentially for transport through the inner membrane.     

Spatial properties of astrocyte gap junction coupling in the rat hippocampus

Gap junction coupling enables astrocytes to form large networks. Its strength determines how easily a signalling molecule diffuses through the network and how far a locally initiated signal can spread. Changes of coupling strength are well-documented during development and in response to various stimuli. Precise quantification of coupling is needed for studying such modifications and their functional consequences. We therefore explored spatial properties of astrocyte coupling in a model simulating dye loading of single astrocytes. Dye spread into the astrocyte network could be characterized by a coupling length constant and coupling anisotropy. In experiments, the fluorescent marker Alexa Fluor 594 was used to measure these parameters in CA1 and dentate gyrus of the rat hippocampus. Coupling did not differ between regions but showed a temperature-dependence, partially owing to changes of intracellular diffusivity, detected by measuring coupling length constants but not the more variable cell counts of dye-coupled astrocytes. We further found that coupling is anisotropic depending on distance to the pyramidal cell layer, which correlated with regional differences of astrocyte morphology. This demonstrates that applying these new analytical approaches provides useful quantitative information on gap junction coupling and its heterogeneity.     

Interdependent Nucleocytoplasmic Trafficking and Interactions of Dis3 with Rrp6, the Core Exosome and Importin‐α3

Subcellular compartmentalization of exoribonucleases (RNAses) is an important control mechanism in the temporal and spatial regulation of RNA processing and decay. Despite much progress towards understanding RNAse substrates and functions, we know little of how RNAses are transported and assembled into functional, subcellularly restricted complexes. To gain insight into this issue, we are studying the exosome‐binding protein Dis3, a processive 3′ to 5′ exoribonuclease. Here, we examine the interactions and subcellular localization of the Drosophila melanogaster Dis3 (dDis3) protein. N‐terminal domain mutants of dDis3 abolish associations with the ‘core’ exosome, yet only reduce binding to the ‘nuclear’ exosome‐associated factor dRrp6. We show that nuclear localization of dDis3 requires a C‐terminal classic nuclear localization signal (NLS). Consistent with this, dDis3 specifically co‐precipitates the NLS‐binding protein importin‐α3. Surprisingly, dDis3 constructs that lack or mutate the C‐terminal NLS retain importin‐α3 binding, suggesting that the interaction is indirect. Finally, we find that endogenous dDis3 and dRrp6 exhibit coordinated nuclear enrichment or exclusion, suggesting that dDis3, Rrp6 and importin‐α3 interact in a complex independent of the core. We propose that the movement and deposition of this complex is important for the subcellular compartmentalization and regulation of the exosome core.     

IL-5 Induces Proliferation and Activation of Microglia via an Unknown Receptor

While the effects of interleukin-3 (IL-3) and granulocyte macrophage-colony stimulating factor (GM-CSF) on microglia are well documented, very little is known about the effects of a related cytokine, interleukin-5 (IL-5). We therefore undertook studies to determine how IL-5 alters various aspects of microglial functioning. Treatment of microglia with IL-5 resulted in the induction of proliferation at levels similar to those induced by GM-CSF. IL-5 also increased cellular metabolism of microglial cells. To determine whether increased metabolism correlated with activation of microglia, we measured levels of nitrite, a breakdown product of nitric oxide. Treatment of microglial cultures with IL-5 increased nitrite levels, while GM-CSF treatment had no effect. Treatment of microglia with IL-5 did not cause activation of the signal transduction pathways linked to the classical IL-5 receptor, STAT5A/5B and ERK1 and ERK2. It is therefore likely that the effects of IL-5 on microglia are not mediated via the classical IL-5 receptor, but rather via a novel receptor.     

Sequence variation in NPC1L1 and association with improved LDL-cholesterol lowering in response to ezetimibe treatment

Niemann-Pick C1-like 1 (NPC1L1) is an intestinal cholesterol transporter and the molecular target of ezetimibe, a cholesterol absorption inhibitor demonstrated to reduce LDL-cholesterol (LDL-C) both as monotherapy and when co-administered with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). Interestingly, significant interindividual variability has been observed for rates of intestinal cholesterol absorption and LDL-C reductions at both baseline and post ezetimibe treatment. To test the hypothesis that genetic variation in NPC1L1 could influence the LDL-C response to ezetimibe, we performed extensive resequencing of the gene in 375 apparently healthy individuals and genotyped hypercholesterolemic patients from clinical trial cohorts. No association was observed between NPC1L1 single-nucleotide polymorphism and baseline cholesterol. However, significant associations to LDL-C response to treatment with ezetimibe were observed in patients treated with ezetimibe in two large clinical trials. Our data demonstrate that DNA sequence variants in NPC1L1 are associated with an improvement in response to ezetimibe pharmacotherapy and suggest that detailed analysis of genetic variability in clinical trial cohorts can lead to improved understanding of factors contributing to variable drug response.     

Synthesis of Non-Natural Pyrimidine Nucleosides

Abstract

Two new non-natural nucleosides bearing an amide (8) or an amidine (9) function have been synthesized. Their properties and the geometry of the exocyclic double bond have been studied.