Extracellular Vesicles in Mycobacterial Infections: Their Potential as Molecule Transfer Vectors.

Extracellular vesicles are membrane-bound structures released by living cells and present in body fluids. Their composition includes proteins, lipids, carbohydrates, and nucleic acids and are involved in transfers between cells. Extracellular vesicles can deliver molecules to cells and tissues even if distant.

Comparative proteomic analysis of spermatozoa isolated by swim-up or density gradient centrifugation.

Background: Reports about the morphologic and functional characteristics of spermatozoa prepared by density gradient centrifugation (DC) or swim-up (SU) have produced discordant results. We have performed a proteomic comparison of cells prepared by DC and SU providing a molecular insight into the differences between these two methods of sperm cell isolation.

Methods: Protein maps were obtained by 2-dimensional (2-D) separations consisting of isoelectrofocusing (IEF) from pI 3 to 11 followed by SDS-polyacrylamide gel electrophoresis.

Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men.

A comparative proteomic study of oligoasthenozoospermic and normozoospermic seminal plasmas was conducted to establish differences in protein expression. Oligoasthenozoospermia (when semen presents with a low concentration and reduced motility of spermatozoa) is common in male infertility. Two-dimensional protein maps from seminal plasma samples from 10 men with normozoospermia and 10 men with idiopathic oligoasthenozoospermia were obtained by isoelectric focusing followed by sodium dodecyl-sulphate polyacrylamide electrophoresis.

An extragranular compartment of blood eosinophils contains eosinophil protein X/eosinophil-derived neurotoxin (EPX/EDN).

Serum and plasma profiles of eosinophil protein X (EPX/EDN) and those of other eosinophil proteins differ in various conditions, suggesting a different mobilisation from storage granules. This work studied the subcellular localisation of EPX/EDN in non-primed and in vivo primed blood eosinophils from healthy and allergic subjects, during and out of the pollen season. Primed eosinophils contain easily mobilisable secretory proteins.

A proteomic insight into the effects of the immunomodulatory hydroxynaphthoquinone lapachol on activated macrophages.

We report the effect of an immunomodulatory and anti-mycobacterial naphthoquinone, lapachol, on the bi-dimensional patterns of protein expression of toll-like receptor 2 (TLR2)-agonised and IFN-γ-treated THP-1 macrophages. This non-hypothesis driven proteomic analysis intends to shed light on the cellular functions lapachol may be affecting. Proteins of both cytosol and membrane fractions were analysed.

The hydroxy-naphthoquinone lapachol arrests mycobacterial growth and immunomodulates host macrophages.

The present study reports the anti-mycobacterial activity of 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone (lapachol) as well as its influence on macrophage functions. Lapachol (L) did not induce apoptosis/necrosis of THP-1 macrophages at ≤32 μg/mL. Mycobacterium avium liquid growth was arrested by ≥32 μg/mL and intra-macrophage proliferation by ≥16 μg/mL lapachol.

The human eosinophil proteome. Changes induced by birch pollen allergy.

Proteins from human eosinophils were separated bidimensionally and identified by mass spectrometry (336 spots/bands, 98 different proteins). Of these, 24.7% belonged to the cytoskeleton/migration group.

Proteomic analysis of human cervical-vaginal fluids.

The pathophysiology of vaginal conditions is still ill-defined at a molecular level. Because the proteome of the human cervical-vaginal fluid (CVF) has not been reported to date, we undertook the identification of proteins present in the cell-free fraction of these fluids. Proteins were separated bidimensionally (2-D) by isoelectrofocusing (pH 3-11) followed by SDS-polyacrylamide electrophoresis.

Complement receptor 3 binds the Borrelia burgdorferi outer surface proteins OspA and OspB in an iC3b-independent manner.

Persistence of borreliae within the vertebrate host depends on the fate of interactions between the spirochetes and target cells. The present work demonstrates the direct binding of the Borrelia burgdorferi outer surface proteins OspA and OspB to CR3 and that this binding is independent of iC3b.

Determination of a large number of kinase activities using peptide substrates, P81 phosphocellulose paper arrays and phosphor imaging.

To perform phosphoproteomics and signal transduction studies, a number of protein kinase activities and levels must be simultaneously analyzed in different cell samples and correlated with phosphoprotein patterns to obtain conclusions with regard to the regulation of kinase networks. We describe here a miniaturized format of the classical phosphocellulose (P81) paper binding assay with which up to 594 kinase reactions can be simultaneously analyzed. Kinase peptide substrates possessing a minimum of three consecutive basic residues were subjected to phosphorylation in 96-well plates and aliquots of the phosphorylation reactions were spotted on arrays printed on P81 papers.

Priming of eosinophil migration across lung epithelial cell monolayers and upregulation of CD11b/CD18 are elicited by extracellular Ca2+.

In patients with asthma, eosinophils are primed and massively infiltrate lung tissues and migrate across epithelia into airways. Using blocking monoclonal antibodies, we found that eosinophil transmigration across a lung epithelial cell monolayer depended on the functions of alphaMbeta2 integrin CD11b/CD18. To study the role of Ca2+ in eosinophil priming and transepithelial migration, we treated eosinophils with eotaxin or thapsigargin (TG), reagents that increase cytoplasmic free Ca2+ concentrations by receptor- or nonreceptor-mediated mechanisms, respectively.

Mycobacteria-containing phagosomes associate less annexins I, VI, VII and XI, but not II, concomitantly with a diminished phagolysosomal fusion.

We have studied the intracellular localization of annexins I,II, VI, VII, and XI in cells containing latex beads or Mycobacterium avium at different times after ingestion in order to establish whether a correlation existed between the association of annexins to phagosomes and phagolysosomal fusion, since the intracellular survival of mycobacteria is linked to an impairment of phagosome maturation. We demonstrate an important decrease in the levels of association of annexins I, VI, VII and XI, but not II to phagosomes containing either live or killed mycobacteria compared with phagosomes containing inert latex particles. The reduced association of annexins observed was detected only on M.