Mycobacteria are intracellular pathogens that can invade and survive within host macrophages, thereby creating a major health problem worldwide. The molecular mechanisms involved in mycobacterial entry are still poorly characterized. Here we report that cholesterol is essential for uptake of mycobacteria by macrophages. Cholesterol accumulated at the site of mycobacterial entry, and depleting plasma membrane cholesterol specifically inhibited mycobacterial uptake. Cholesterol also mediated the phagosomal association of TACO, a coat protein that prevents degradation of mycobacteria in lysosomes. Thus, by entering host cells at cholesterol-rich domains of the plasma membrane, mycobacteria may ensure their subsequent intracellular survival in TACO-coated phagosomes.

Chemotherapeutic options to treat tuberculosis are severely restricted by the intrinsic resistance of Mycobacterium tuberculosis to the majority of clinically applied antibiotics. Such resistance is partially provided by the low permeability of their unique cell envelope. Here we describe a complementary system that coordinates resistance to drugs that have penetrated the envelope, allowing mycobacteria to tolerate diverse classes of antibiotics that inhibit cytoplasmic targets. This system depends on whiB7, a gene that pathogenic Mycobacterium shares with Streptomyces, a phylogenetically related genus known as the source of diverse antibiotics. In M. tuberculosis, whiB7 is induced by subinhibitory concentrations of antibiotics (erythromycin, tetracycline, and streptomycin) and whiB7 null mutants (Streptomyces and Mycobacterium) are hypersusceptible to antibiotics in vitro. M. tuberculosis is also antibiotic sensitive within a monocyte model system. In addition to antibiotics, whiB7 is induced by exposure to fatty acids that pathogenic Mycobacterium species may accumulate internally or encounter within eukaryotic hosts during infection.Gene expression profiling analyses demonstrate that whiB7 transcription determines drug resistance by activating expression of a regulon including genes involved in ribosomal protection and antibiotic efflux. Components of the whiB7 system may serve as attractive targets for the identification of inhibitors that render M. tuberculosis or multidrug-resistant derivatives more antibioticsensitive.multidrug resistance ͉ Streptomyces ͉ WhiB ͉ microarray ͉ gene expression

Macitentan, also called Actelion-1 or -6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-NЈ-propylaminosulfonamide], is a new dual ET A / ET B endothelin (ET) receptor antagonist designed for tissue targeting. Selection of macitentan was based on inhibitory potency on both ET receptors and optimization of physicochemical properties to achieve high affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major and pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonized the specific binding of ET-1 on membranes of cells overexpressing ET A and ET B receptors and blunted ET-1-induced calcium mobilization in various natural cell lines, with inhibitory constants within the nanomolar range. In functional assays, macitentan and ACT-132577 inhibited ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET A receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea (ET B receptors). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricle hypertrophy, and it markedly improved survival. In diabetic rats, chronic administration of macitentan decreased blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural injury). In conclusion, macitentan, by its tissuetargeting properties and dual antagonism of ET receptors, protects against end-organ damage in diabetes and improves survival in pulmonary hypertensive rats. This profile makes macitentan a new agent to treat cardiovascular disorders associated with chronic tissue ET system activation.

T cell homeostasis is essential for the functioning of the vertebrate immune system, but the intracellular signals required for T cell homeostasis are largely unknown. We here report that the WD-repeat protein family member coronin-1, encoded by the gene Coro1a, is essential in the mouse for T cell survival through its promotion of Ca2+ mobilization from intracellular stores. Upon T cell receptor triggering, coronin-1 was essential for the generation of inositol-1,4,5-trisphosphate from phosphatidylinositol-4,5-bisphosphate. The absence of coronin-1, although it did not affect T cell development, resulted in a profound defect in Ca2+ mobilization, interleukin-2 production, T cell proliferation and T cell survival. We conclude that coronin-1, through activation of Ca2+ release from intracellular stores, is an essential regulator of peripheral lymphocyte survival.

Coronin 1 is a member of the coronin protein family specifically expressed in leukocytes and accumulates at sites of rearrangements of the F-actin cytoskeleton. Here, we describe that coronin 1 molecules are coiled coil-mediated homotrimeric complexes, which associate with the plasma membrane and with the cytoskeleton via two distinct domains. Association with the cytoskeleton was mediated by trimerization of a stretch of positively charged residues within a linker region between the N-terminal, WD repeat-containing domain and the C-terminal coiled coil. In contrast, neither the coiled coil nor the positively charged residues within the linker domain were required for plasma membrane binding, suggesting that the N-terminal, WD repeat-containing domain mediates membrane interaction. The capacity of coronin 1 to link the leukocyte cytoskeleton to the plasma membrane may serve to integrate outside-inside signaling with modulation of the cytoskeleton. INTRODUCTIONCoronin 1 is expressed exclusively by leukocytes (Suzuki et al., 1995;Ferrari et al., 1999) and is a member of the WD repeat protein family termed coronins, which are collectively defined as F-actin-associated proteins widely expressed in the eukaryotic kingdom (de Hostos, 1999). In Dictyostelium discoideum, coronin colocalizes with F-actin filaments at crown-shaped phagocytic cups and macropinosomes (de Hostos et al., 1991(de Hostos et al., , 1993Maniak et al., 1995;Fukui et al., 1999). Dictyostelium deleted for coronin displays strong reduction in cell locomotion, phagocytosis, macropinocytosis, and cytokinesis, indicating that in this slime mold coronin is functionally involved in F-actin-based motility-related processes (de Hostos et al., 1993). In Saccharomyces cerevisiae, the single coronin isoform Crn1p was found to localize to cortical F-actin patches in an actin-dependent manner (Heil-Chapdelaine et al., 1998). In vitro, Crn1p can nucleate and cross-link F-actin filaments and bind to microtubules (Goode et al., 1999). Recently, yeast Crn1 was proposed to promote the formation of actin filament networks based on its interaction with the Arp2/3 complex (Humphries et al., 2002). Interestingly, unlike the Dictyostelium coronin-null mutant, an S. cerevisiae Crn1p-null-mutant does not show any phenotype in actin-dependent processes (Heil-Chapdelaine et al., 1998), suggesting that in this organism coronin does not perform an essential function in regulating the actin cytoskeleton. Although lower eukaryotes have one coronin gene, database searches have revealed the existence of several coronins in humans and mice (denoted coronins 1-7) (Okumura et al., 1998;de Hostos, 1999;Rybakin et al., 2004).In macrophages and lymphocytes, coronin 1 concentrates at sites of rearrangement of the cytoskeleton. In lymphocytes, coronin 1 assembles at the immunological synapse formed during activation of T-cells (Nal et al., 2004). In mouse macrophages, coronin 1 accumulates during phagocytosis at the cytosolic face of phagosomes and is actively retained by pathogenic mycoba...