Rodolfo Garcia

Altered intracellular redox status in Gaucher disease fibroblasts and impairment of adaptive response against oxidative stress

Gaucher disease (GD) is a lysosomal storage disorder, due to glucosylceramide (GlcCer) accumulation in several body tissues, which causes cellular failure by yet unidentified mechanisms. Several evidence indicates that GD pathogenesis is associated to an impairment in intracellular redox state. In fibroblast primary cultures, reactive oxygen species (ROS) levels and protein carbonyl content resulted significantly increased in GD patients compared to healthy donors, suggesting that GD cells, facing a condition of chronic oxidative stress, have evolved an adaptive response to survive. The ROS rise is probably due to NAD(P)H oxidase activity, being inhibited by the treatment with diphenylene iodonium chloride. Interestingly, GD cells are more sensitive to H2O2 induced cell death, suggesting a dysregulation in the adaptive response to oxidative stress in which APE1/Ref‐1 plays a central role. We found that the cytoplasmic amounts of APE1/Ref‐1 protein were significantly higher in GD fibroblasts with respect to controls, and that GD cells failed to upregulate its expression upon H2O2 treatment. Both ROS and APE1/Ref‐1 increases are due to GlcCer accumulation, being prevented by treatment of GD fibroblasts with Cerezyme® and induced in healthy fibroblasts treated with conduritol‐β‐epoxide. These data, suggesting that GD cells display an impairment in the cellular redox state and in the adaptive cellular response to oxidative stress, may open new perspectives in the comprehension of GD pathogenesis. J. Cell. Physiol. 212: 223–235, 2007.

Eosinophil cationic protein is stored in, but not produced by, peripheral blood neutrophils

Background Eosinophil cationic protein (ECP) is an eosinophil‐derived protein, which has been shown to be present in circulating neutrophils.

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

ABSTRACT 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.

Leptospires are killed in vitro by both oxygen-dependent and -independent reactions.

ABSTRACT This study reports for the first time that leptospires are killed by H2O2 and by low-molecular-weight primary granule components, which are agents normally released by neutrophils upon stimulation. Although both pathogenic and nonpathogenic strains were sensitive to H2O2-mediated killing, nonpathogenic organisms were found to be more susceptible. In addition, the killing of leptospires by H2O2 was found to be independent of the presence of the neutrophil primary granule component myeloperoxidase and therefore not a consequence of halogenation reactions. We have also determined that leptospires are significantly sensitive only to primary granule components and, among those, to proteins and/or peptides of less than 30 kDa.

IL‐5 priming of the PMA‐induced oxidative metabolism of human eosinophils from allergic and normal subjects during a pollen season

Aim To study the effect of IL‐5 priming on the PMA‐induced oxidative metabolism of blood eosinophils from allergic patients and healthy controls, during pollen exposure.

An update on the role of miRNA-155 in pathogenic microbial infections.

MicroRNAs (miRNAs) are evolutionarily conserved and naturally abundant molecules of single-stranded, non-coding RNA from ∼17 to 25 nucleotides long. MiRNAs act at post-transcriptional level either to suppress gene translation or to induce mRNA degradation, according to the degree of complementarity with their target sequences. MiR-155 is a typical representative of the miRNA family that plays a crucial role in cell differentiation and organism development. A number of studies have shown that miR-155 can not only regulate cell proliferation, apoptosis and lymphoma progression, but also plays an important part in various other physiological and pathological processes. For instance, it is involved in hematopoietic cell differentiation, cardiovascular disease, inflammation and immune responses. In recent years, the role of miR-155 in infectious diseases has attracted considerable attention. This review will highlight the participation of miR-155 in the responses to infections caused by different pathogens.

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. avium-containing phagosomes and not in other cell membrane nor in cytosolic fractions from infected cells, and was apparent from 8 hours through to 4 days after phagocytosis. These findings add elements to the present knowledge of the phagosomal modifications that accompany the survival of intracellular pathogens, suggesting that annexins I, VI, VII, and XI play a secondary role in phagosomal fusion events while annexin II does not seem to be related to the mechanism of regulation of endolysosomal fusion.

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. Phosphorylation levels were quantified using a storage phosphor system imager. The versatility of the procedure was validated by analyzing casein kinase 2, protein kinase C, and p34cdc2/cyclin B in cell extracts and testing the effect of known inhibitors and activators on kinase activities. This improved, miniaturized version of the classical P81 paper method combines simplicity, high sensitivity, high reproducibility, high reliability, and optimal Z factors and takes into account possible sources of background signals. We discuss the possibility of automation and the advantages over other methods.

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. Map images were analysed using dedicated software involving normalization, spot-to-spot volume comparison and statistical treatment of the results to establish the significance of differences between normal and oligoasthenozoospermic samples. Six out of 1028 spots showed over 1.5-fold relative intensity differences (P < 0.05, analysis of variance). Four proteins were identified by nano liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry of their tryptic peptides and database searches. Two proteins were more than three-fold under-expressed in oligoasthenozoospermia, namely epididymal secretory protein E1 and galectin-3-binding protein; the other (lipocalin-1 and a prolactin-inducible protein form) were over-expressed. The identity and differential expression of epididymal secretory protein E1 was verified by Western-blotting. The statistically significant differential expression of these four proteins in oligoasthenozoospermia compared with normozoospermia provides a molecular basis for further investigations into the pathogenic mechanisms underlying idiopathic oligoasthenozoospermia.

L273S missense substitution in human lysosomal acid lipase creates a new N-glycosylation site

Human lysosomal acid lipase (LAL), when expressed in Hela cells using the Vaccinia T7 expression system, showed four major molecular forms ranging from 42 to 54 kDa. Treatment with endoglycosidase H resulted in a 42 kDa protein, indicating that the molecular weight variations were due to N‐glycosylation. A missense substitution, L273S, previously detected in a patient with cholesteryl ester storage disease (CESD), produced catalytically inactive LAL showing a largest molecular mass form of 56 kDa instead of 54 kDa. Analysis of the amino acid sequence in the close proximity of the mutation (NMS → NML) indicated that the L273S mutation creates an additional N‐glycosylation consensus (N‐X‐S/T) in this region. Two site directed mutants disrupting this consensus, QMS and QML, when expressed in HeLa cells, did not show the 56 kDa form but the normal 54 kDa band whereas deglycosylation always resulted in the major 42 kDa form, as observed with normal LAL and the L273S mutant. These data confirmed that an additional N‐glycosylation at N271 was responsible for the 56 kDa form of the protein produced from the L273S allele. Furthermore, deglycosylation of normal LAL reduced the acid hydrolase activity towards both tri‐oleyl glycerol and cholesteryl oleate by 50%, strongly suggesting that N‐linked carbohydrate residues are important for optimal catalytic activity.