Paraoxonase1-192 polymorphism modulates the effects of regular and acute exercise on paraoxonase1 activity

Regular exercise practise is a protective factor against coronary heart disease and enhances antioxidant systems, whereas acute exercise appears to be a major source of increased oxidative stress. Paraoxonase1 (PON1) is an antioxidant HDL-linked enzyme, whose activity toward paraoxon (PON1 activity) is strongly modulated by the PON1-192 polymorphism, comprising Q and R alleles for low and high PON1 activity, respectively. Another polymorphism at the PON1 locus, the PON1-55, modulates PON1 protein and activity levels. PON1 activity, lipid levels, and oxidized LDL concentration were determined in 17 healthy young volunteers before and after a 16-weeks aerobic exercise training period. Furthermore, PON1 activity was analyzed after a bout of exercise in both situations. We found that regular exercise was associated with a decrease in oxidized LDL levels, and an increase in PON1 activity in QQ subjects and with a decrease in PON1 activity in R carriers. A bout of exercise produced an increase in PON1 activity just after the bout of exercise, followed by a decrease in its activity. A recovery of the basal PON1 activity levels at 24 h was found in QQ subjects regardless of their training status and in trained R carriers, but not in untrained R carriers. These results suggest that the effects of regular and acute exercise on PON1 activity levels are modulated by PON1-192 polymorphism. Changes were less evident for the PON1-55 polymorphism.     

The peptide repertoires of HLA-B27 subtypes differentially associated to spondyloarthropathy (B*2704 and B*2706) differ by specific changes at three anchor positions

HLA-B*2704 is strongly associated with ankylosing spondylitis. B*2706, which differs from B*2704 by two amino acid changes, is not associated with this disease. A systematic comparison of the B*2704- and B*2706-bound peptide repertoires was carried out to elucidate their overlap and differential features and to correlate them with disease susceptibility. Both subtypes shared about 90% of their peptide repertoires, consisting of peptides with Arg(2) and C-terminal aliphatic or Phe residues. B*2706 polymorphism influenced specificity at three anchor positions: it favored basic residues at P3 and POmega-2 and impaired binding of Tyr and Arg at POmega. Thus, the main structural feature of peptides differentially bound to B*2704 was the presence of C-terminal Tyr or Arg, together with a strong preference for aliphatic/aromatic P3 residues. This is the only known feature of B*2704 and B*2706 that correlates to their differential association with spondyloarthropathy. The concomitant presence of basic P3 and POmega-2 residues was observed only among peptides differentially bound to B*2706, suggesting that it impairs binding to B*2704. Similarity between peptide overlap and the degree of cross-reaction with alloreactive T lymphocytes suggested that the majority of shared ligands maintain unaltered antigenic features in the context of both subtypes.     

Ontogenesis of the vitamin D receptor in rat heart

1,25-Dihydroxyvitamin D(3) through its receptor (vitamin D receptor; VDR) has important physiological effects such as calcium transport and cell growth and differentiation. Although the VDR is present in a variety of cell lines as well as in numerous tissues, including rat and human heart, no data are available about the presence of VDR in heart at different steps of rat life. In this study we evaluated the VDR expression using RT-PCR and immunohistochemical techniques in fetal (17, 18 and 20 gestational days), neonatal (4 and 8 days) and adult rat heart. Immunohistochemical techniques showed the VDR protein localisation in the nuclei of cardiac muscle fibres. Also, we demonstrated that VDR mRNA expression is changing over these different periods of development, showing significant differences in 20 days versus 18 days of fetal age. These changes in VDR expression may be related to other parameters associated with the development of the cardiac muscle and/or intracellular cardiac cell calcium homeostasis.     

In vivo studies on mouse erythrocytes linked to transferrin

Transferrin (Tf), a plasma protein with numerous, highly specific receptors in proliferating and differentiating cells was already discussed as a targeting ligand for drugs and liposomes in previous studies. In this paper, we deal with erythrocytes linked to Tf as possible physiological targeting carrier systems for delivering anticancer drugs. For that purpose we have used glutaraldehyde (0.1%) as a coupling agent between Tf and erythrocytes. The highest amount of Tf linked to erythrocytes turned out to be 0.76 +/- 0.13 microg Tf/10(6) cells, while reaching 65% of cell recovery. After 13 days, the Tf-erythrocytes hemolysis reached 50%, with transferrin still coupled to erythrocytes. The in vivo kinetic behaviour of intravenously injected 51Cr-Tf-erythrocytes showed a reduced half-life to hours as compared to days of controls. However, a considerable percentage of Tf-erythrocytes (close to 20%) remained circulating for a relatively long period (around 2 days), which made possible the specific targeting by these carrier systems. In vivo biodistribution studies indicated that 51Cr-Tf-erythrocytes rapidly accumulated in the different studied organs (liver, spleen, lungs, kidneys, femur-tibia, and heart), suggesting a selective removal of Tf-erythrocytes by the cells of the mononuclear phagocytic system present mainly in liver and spleen. On the other hand, Tf-erythrocytes showed a poor targeting of heart tissue, therefore a reduced cardiac toxicity should be expected after administration of erythrocyte-encapsulated drugs. The presence of Tf-erythrocytes in femur-tibia and spleen could be related to the Tf-specific binding to the hematopoietic cells containing Tf receptors. The final results of this study encourage additional research on Tf-erythrocyte to investigate the relationship between transferrin-mediated targeting by carrier erythrocytes and uptake of different erythrocyte-encapsulated drugs. Consequently, the current study showed possible use of these carriers as a potential therapeutic tool for drug targeting in animal models with alterations affecting mononuclear phagocytic system or carcinomas of various origins whose cells show elevated number of Tf receptors.     

Identification of p21Cip1 binding proteins by gel electrophoresis and capillary liquid chromatography microelectrospray tandem mass spectrometry

Proteins bound to a glutathione-S-transferase-p21Cip1 affinity column were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using tandem mass spectrometry. Capillary liquid chromatography coupled to microelectrospray tandem mass spectrometry (capLC-microESI MS/MS) in an ion trap allowed identification of the proteins present in the gel bands. Of eleven bands analyzed, fifty-three proteins were identified. More than one hundred tryptic peptides were detected on-line, automatically fragmented and used for protein characterization in databases. Samples were also analyzed by off-line nanospray and matrix-assisted laser desorption/ionization mass spectrometry. CapLC-microESI MS/MS was the most efficient technique for the analysis of these protein mixtures.     

Interleukin-dependent modulation of HLA-DR expression on CD4and CD8 activated T cells

Summary Interleukins (IL) regulate different T-cell surface Ag known as activation markers that have distinct functional roles. In this paper, while studying the influence of some cytokines(IL-12, IL-2 and IL-4) on the expression of several markers [CD69,CD25, CD26, CD3, human leukocyte antigen (HLA-DR), CD45R0] in in vitro activated human T lymphocytes, we observed two groups of donors responding to phytohaemagglutinin (PHA) activation with high or low HLA-DRAg expression. We also found that CD4 and CD8 populations had different HLA-DR densities under PHA activation (particularly the high HLA-DR-expressing group). Interleukins, in a dose-dependent manner (IL-2 partially),upregulated these HLA-DR levels. In 5 day cultures, IL-12 and IL-2 enhanced the CD8/CD4 ratio of activated T cells,which was responsible, in part, for the IL-dependent HLA-DR upregulation.IL-12 and IL-2 also upregulated the HLA-DR expression at the molecular level on CD8, and IL-12 downregulated it on CD4 cells. It seems that IL-4 upregulated HLA-DR by shortening the mitogen-dependent regulation kinetics. We hypothesize that the different effect of each IL on HLA-DR expression might be related to the regulation of the dose of antigenic peptide presentation and, thus, also influence TH1/TH2 dominance.     

Aspergillus galactomannan detection in allogenic hematopoietic cell transplantation

Invasive aspergillosis has become the leading cause of death after allogeneic hematopoietic stem cell transplantation. This is partially due to the lack of a prompt diagnosis. Recently the detection of Aspergillus galactomannan antigen by means an ELISA technique in serum has been described. The objective of this study was to validate its usefulness in the allogeneic hematopoietic stem cell transplantation setting.