Nanoparticles (microvesicles – MVs) are found in peripheral blood. They bud from cell membranes. During the isolation process additional MVs are released from blood cells, so optimization and reduction of additional MVs are pivotal if MVs are used as a diagnostic marker for certain diseases. We showed that a size and a concentration of MVs are dependent on temperature during the isolation, where higher concentrations of smaller MVs were observed at lower temperatures. On the basis of pictures of deformed blood cells found in the isolates indicate we can speculate how fragmentation of blood cells may take place. The results show that the contents of isolates reflect the properties of blood cells and their interaction with the surrounding solution, which differ in different diseasesand may therefore present a relevant clinical parameter.
COBISS.SI-ID: 29065433
Lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.
COBISS.SI-ID: 4957722
Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures.
COBISS.SI-ID: 4824602
Myristoylated alanine-rich C kinase substrate (MARCKS) is an intrinsically unfolded protein, which mediates the cross-talk between several signal transduction pathways. Transcription of MARCKS is increased by stimulation with bacterial LPS. We determined that MARCKS specifically binds to LPS. After LPS stimulation, MARCKS moved from the plasma membrane to FYVE-positive endosomes, where it colocalized with LPS. MARCKS-deficient mouse embryonic fibroblasts (MEFs) responded to LPS with increased IL-6 production compared with the matched wild-type MEFs. Similarly, small interfering RNA knockdown of MARCKS also increased LPS signaling, whereas overexpression of MARCKS inhibited LPS signaling. These findings demonstrate that MARCKS contributes to the negative regulation of the cellular response to LPS.
COBISS.SI-ID: 4930586
TLR4 is involved in activation of innate immune response. Despite numerous studies, the role of separate domains of TLR4 in the regulation of receptor activation is poorly understood. Replacement of the TLR4 ectodomain with LPS-binding proteins MD-2 or CD14 resulted in a robust ligand-independent constitutive activation. This demonstratesan intrinsic dimerization propensity of the transmembrane and cytoplasmic domains of TLR4 and reveals a previously unknown function of the ectodomain in inhibiting spontaneous receptor dimerization. This is important for the sensitivity of TLR4 to activation by different agonists.
COBISS.SI-ID: 4652570