Phthiocerol dimycocerosates of M. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids.

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.

Membrane partitioning of various delta-opioid receptor forms before and after agonist activations: the effect of cholesterol.

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR*) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR* is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR* was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.

Changes of the membrane lipid organization characterized by means of a new cholesterol-pyrene probe.

We synthesized 3beta-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): K(DOPC) >> K(POPC) >> K(DMPC) > K(DMPC/15 mol % Chol) > K(DMPC/30 mol % Chol). This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I(1)-I(5)) and excimer (I(E)) emission bands. In model membranes, I(1)/I(3) and I(E)/I(3) ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse delta-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse delta-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.

The dose-dependent toxicological effects and potential perturbation on the neurotransmitter secretion in brain following intranasal instillation of copper nanoparticles.

Increasing production and application of metallic nanomaterials are likely to result in the release of these particles into the environment. These released nanoparticles may enter into the lungs and the central nervous system (CNS) directly through inhalation, which therefore poses a potential risk to human health. Herein, we focus on the systemic toxicity and potential influence on the neurotransmitter secretion of intranasally instilled copper nanoparticles (23.5 nm) at three different doses. Copper nanoparticle-exposed mice exhibit pathological lesions at different degrees in certain tissues and especially in lung tissue as revealed by histopathology and transmission electron microscopy (TEM) observations. Inductively-coupled plasma mass spectrometry (ICP-MS) results show that the liver, lung and olfactory bulb are the main tissues in which the copper concentrations increased significantly after exposure to a higher level of Cu nanoparticles (40 mg/kg of body weight). The secretion levels of various neurotransmitters changed as well in some brain regions, especially in the olfactory bulb. Our results indicate that the intranasally instilled copper nanoparticles not only cause the lesions where the copper accumulates, but also affect the neurotransmitter levels in the brain.

The triggering of apoptosis in macrophages by pristine graphene through the MAPK and TGF-beta signaling pathways.

With the development of nanotechnology and the wide use of graphene, it has become necessary to assess the potential biological adverse effects of graphene. However, most of the recent publications are focused on various modified graphenes. We demonstrated biological effects of commercial pristine graphene in murine RAW 264.7 macrophages, which is an important effector cells of the innate immune system. We found that the pristine graphene can induce cytotoxicity through the depletion of the mitochondrial membrane potential (MMP) and the increase of intracellular reactive oxygen species (ROS), then trigger apoptosis by activation of the mitochondrial pathway. The MAPKs (JNK, ERK and p38) as well as the TGF-beta-related signaling pathways were found to be activated in the pristine grapheme-treated cells, which activated Bim and Bax, two pro-apoptotic member of Bcl-2 protein family. Consequently, the caspase 3 and its downstream effector proteins such as PARP were activated and the execution of apoptosis was initiated. This study provides an insight for the suppression of the apoptosis induced by the graphene through the mitochondrial pathways, the MAPKs- and TGF-beta-related signaling pathways.

Rutile TiO2 particles exert size and surface coating dependent retention and lesions on the murine brain.

The rising commercial use and large-scale production of engineered nanoparticles (NPs) may lead to unintended exposure to humans. The central nervous system (CNS) is a potential susceptible target of the inhaled NPs, but so far the amount of studies on this aspect is limited. Here, we focus on the potential neurological lesion in the brain induced by the intranasally instilled titanium dioxide (TiO2) particles in rutile phase and of various sizes and surface coatings. Female mice were intranasally instilled with four different types of TiO2 particles (i.e. two types of hydrophobic particles in micro- and nano-sized without coating and two types of water-soluble hydrophilic nano-sized particles with silica surface coating) every other day for 30 days. Inductively coupled plasma mass spectrometry (ICP-MS) were used to determine the titanium contents in the sub-brain regions. Then, the pathological examination of brain tissues and measurements of the monoamine neurotransmitter levels in the sub-brain regions were performed. We found significant up-regulation of Ti contents in the cerebral cortex and striatum after intranasal instillation of hydrophilic TiO2 NPs. Moreover, TiO2 NPs exposure, in particular the hydrophilic NPs, caused obvious morphological changes of neurons in the cerebral cortex and significant disturbance of the monoamine neurotransmitter levels in the sub-brain regions studied. Thus, our results indicate that the surface modification of the NPs plays an important role on their effects on the brain. In addition, the difference in neurotoxicity of the two types of hydrophilic NPs may be induced by the shape differences of the materials. The present results suggest that physicochemical properties like size, shape and surface modification of the nanomaterials should be considered when evaluating their neurological effects.

Pulmonary responses to printer toner particles in mice after intratracheal instillation.

The release of ultrafine particles from office equipment is currently receiving great concerns due to its potential threat to human health when inhaled. Printer toner is one of the largest consumables in daily office work, and the particles released from printers and photocopiers may pose damage to respiratory system. In this study, we found the particles can be released into the surrounding environment during the printing process and the concentrations of PM(2.5) and PM(10) particles increased obviously. To evaluate the time-course pulmonary responses caused by toner particles, the toner suspension was instilled into the lungs of the male mice through intratracheally instillation every other day for four times and the pulmonary responses of the lung were monitored at days 9, 28, 56 and 84. Indeed, mice treated with toner particles displayed a slower body weight growth rate during the recovery phase. The total cell number in bronchoalveolar lavage fluids (BALF) of toner-exposed groups was much higher than the saline-treated groups. The total protein, lactate dehydrogenase and acid phosphatase in BALF exhibited significant changes (p<0.05 or p<0.01) at different time points. The nitric oxide synthase, interleukin 1-beta, and interleukin 6 in the lung tissue of the toner-exposed groups also exhibited significant changes (p<0.05 or p<0.01). The pathological examination showed that toner particles can adhere to the alveolar septal walls, then enter into the alveoli and cause pulmonary lesion. During the experimental period, particles phagocytosed by alveolar macrophages (AMs) led to an increase of both AMs number and apoptosis. The pulmonary stress still remained over time even with a clearance period for 12 weeks. These results indicate that exposure to toner particles can inhibit the normal growth of the mice and induce significant inflammatory responses and lesion in the lung tissues. The health and safety effects from working indoors in offices with fumes and particles released from photocopiers and printers need to be paid more attention.