The outer membrane of Brucella ovis shows increased permeability to hydrophobic probes and is more susceptible to cationic peptides than are the outer membranes of mutant rough Brucella abortus strains

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-L-lysine, and poly-L-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells

Multiple Phenotypes in Adult Mice following Inactivation of the Coxsackievirus and Adenovirus Receptor (Car) Gene

To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo

Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents

Purpose: In future therapeutics new formulas are needed that assure lower doses, fewer side effects, targeted administration and protection of the drug from degradation. In a first step to fulfil the requirements defined above, we carried out an in vitro study by developing a new procedure to encapsulate drugs using native vesicles first from prostasomes and then from erythrocyte membranes known to be well tolerated. The new method for production of drug delivery vesicles utilized osmotic loading of detergent resistant membranes (DRMs). Materials and methods: DRMs of prostasomes and prepared human erythrocyte membranes were extracted and separated in a sucrose gradient at a density of 1.10 g/mL containing 1% Triton X-100. These DRMs were characterized by electron microscopy (transmission and scanning EM) and loaded with low and high molecular compounds. PC3 prostate cancer cells were treated with doxorubicin loaded DRMs in triplicate. DAPI (nuclear fluorescent stain) was included and fluorescence microscopic pictures were taken before the cells were trypsinized and counted after 48h. Results: The content of the well separated band was observed ultrastructurally as small spherical, double layered membrane vesicles, (DRM vesicles) which harbored hyperosmolar sucrose of the gradient. Encapsulated hyperosmolar sucrose induced a transient osmotic lysis of the DRM vesicles when suspended in isotonic buffer containing loading molecules allowing vesicular inclusion. After this proof of concept, the method was finally employed for doxorubicin loading of DRM vesicles from human erythrocytes. When incubating such vesicles with PC3 cells a complete arrest of growth was observed in sharp contrast to PC3 cells incubated with plain doxorubicin in similar conditions. Conclusion: The present results open up new possibilities for using DRM vesicles as drug delivery vesicles.Louise Dubois and Liza Löf contributed equally to this work.</p

Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents

Purpose: In future therapeutics new formulas are needed that assure lower doses, fewer side effects, targeted administration and protection of the drug from degradation. In a first step to fulfil the requirements defined above, we carried out an in vitro study by developing a new procedure to encapsulate drugs using native vesicles first from prostasomes and then from erythrocyte membranes known to be well tolerated. The new method for production of drug delivery vesicles utilized osmotic loading of detergent resistant membranes (DRMs). Materials and methods: DRMs of prostasomes and prepared human erythrocyte membranes were extracted and separated in a sucrose gradient at a density of 1.10 g/mL containing 1% Triton X-100. These DRMs were characterized by electron microscopy (transmission and scanning EM) and loaded with low and high molecular compounds. PC3 prostate cancer cells were treated with doxorubicin loaded DRMs in triplicate. DAPI (nuclear fluorescent stain) was included and fluorescence microscopic pictures were taken before the cells were trypsinized and counted after 48h. Results: The content of the well separated band was observed ultrastructurally as small spherical, double layered membrane vesicles, (DRM vesicles) which harbored hyperosmolar sucrose of the gradient. Encapsulated hyperosmolar sucrose induced a transient osmotic lysis of the DRM vesicles when suspended in isotonic buffer containing loading molecules allowing vesicular inclusion. After this proof of concept, the method was finally employed for doxorubicin loading of DRM vesicles from human erythrocytes. When incubating such vesicles with PC3 cells a complete arrest of growth was observed in sharp contrast to PC3 cells incubated with plain doxorubicin in similar conditions. Conclusion: The present results open up new possibilities for using DRM vesicles as drug delivery vesicles.<p>Louise Dubois and Liza Löf contributed equally to this work.</p

The outer membrane of Brucella ovis shows increased permeability to hydrophobic probes and is more susceptible to cationic peptides than are the outer membranes of mutant rough Brucella abortus strains

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-L-lysine, and poly-L-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells

MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cells

The MYC genes are the most frequently activated oncogenes in human tumors and are hence attractive therapeutic targets. MYCN amplification leads to poor clinical outcome in childhood neuroblastoma, yet strategies to modulate the function of MYCN do not exist. Here we show that 10058-F4, a characterized c-MYC/Max inhibitor, also targets the MYCN/Max interaction, leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-amplified neuroblastoma cells and to increased survival of MYCN transgenic mice. We also report the discovery that inhibition of MYC is accompanied by accumulation of intracellular lipid droplets in tumor cells as a direct consequence of mitochondrial dysfunction. This study expands on the current knowledge of how MYC proteins control the metabolic reprogramming of cancer cells, especially highlighting lipid metabolism and the respiratory chain as important pathways involved in neuroblastoma pathogenesis. Together our data support direct MYC inhibition as a promising strategy for the treatment of MYC-driven tumors

Mitochondrial fusion is required for regulation of mitochondrial DNA replication.

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA