Generation of CD4(+)CD45RA(+) effector T cells by stimulation in the presence of cyclic adenosine 5'-monophosphate-elevating agents

After TCR cross-linking, naive CD4(+)CD45RA(+) T cells switch to the expression of the CD45RO isoform and acquire effector functions. In this study we have shown that cAMP-elevating agents added to anti-CD3- and anti-CD28-stimulated cultures of T lymphocytes prevent acquisition of the CD45RO(+) phenotype and lead to the generation of a new subpopulation of primed CD4(+)CD45RA(+) effector cells (cAMP-primed CD45RA). These cells displayed a low apoptotic index, as the presence of dibutyryl cAMP (dbcAMP)-rescued cells from CD3/CD28 induced apoptosis. Inhibition of CD45 splicing by dbcAMP was not reverted by addition of exogenous IL-2. cAMP-primed CD45RA cells had a phenotype characteristic of memory/effector T lymphocytes, as they showed an up-regulated expression of CD2, CD44, and CD11a molecules, while the levels of CD62L Ag were down-regulated. These cells also expressed the activation markers CD30, CD71, and HLA class II Ags at an even higher level than CD3/CD28-stimulated cells in the absence of dbcAMP. In agreement with this finding, cAMP-primed CD45RA cells were very efficient in triggering allogenic responses in a MLR. In addition, cAMP-primed CD45RA cells produce considerable amounts of the Th2 cytokines, IL-4, IL-10, and IL-13, whereas the production of IFN-gamma and TNF-alpha was nearly undetectable. The elevated production of IL-13 by neonatal and adult cAMP-primed CD45RA cells was specially noticeable. The cAMP-dependent inhibition of CD45 splicing was not caused by the production of immunosuppressor cytokines. These results suggest that within the pool of CD4(+)CD45RA(+) cells there is a subpopulation of effector lymphocytes generated by activation in the presence of cAMP-elevating agents.     

Suicide prevention: disruption of apoptotic pathways by protozoan parasites

The modulation of apoptosis has emerged as an important weapon in the pathogenic arsenal of multiple intracellular protozoan parasites. Cryptosporidium parvum, Leishmania spp., Trypanosoma cruzi, Theileria spp., Toxoplasma gondii and Plasmodium spp. have all been shown to inhibit the apoptotic response of their host cell. While the pathogen mediators responsible for this modulation are unknown, the parasites are interacting with multiple apoptotic regulatory systems to render their host cell refractory to apoptosis during critical phases of intracellular infection, including parasite invasion, establishment and replication. Additionally, emerging evidence suggests that the parasite life cycle stage impacts the modulation of apoptosis and possibly parasite differentiation. Dissection of the host-pathogen interactions involved in modulating apoptosis reveals a dynamic and complex interaction that recent studies are beginning to unravel.     

S-nitrosoglutathione reductase affords protection against pathogens in Arabidopsis, both locally and systemically

Nitric oxide and S-nitrosothiols (SNOs) are widespread signaling molecules that regulate immunity in animals and plants. Levels of SNOs in vivo are controlled by nitric oxide synthesis (which in plants is achieved by different routes) and by S-nitrosoglutathione turnover, which is mainly performed by the S-nitrosoglutathione reductase (GSNOR). GSNOR is encoded by a single-copy gene in Arabidopsis (Arabidopsis thaliana; Martínez et al., 1996; Sakamoto et al., 2002). We report here that transgenic plants with decreased amounts of GSNOR (using antisense strategy) show enhanced basal resistance against Peronospora parasitica Noco2 (oomycete), which correlates with higher levels of intracellular SNOs and constitutive activation of the pathogenesis-related gene, PR-1. Moreover, systemic acquired resistance is impaired in plants overexpressing GSNOR and enhanced in the antisense plants, and this correlates with changes in the SNO content both in local and systemic leaves. We also show that GSNOR is localized in the phloem and, thus, could regulate systemic acquired resistance signal transport through the vascular system. Our data corroborate the data from other authors that GSNOR controls SNO in vivo levels, and shows that SNO content positively influences plant basal resistance and resistance-gene-mediated resistance as well. These data highlight GSNOR as an important and widely utilized component of resistance protein signaling networks conserved in animals and plants.     

Pulmonary surfactant protein A inhibits the lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria and microsomes

Reactive oxygen species play an important role in several acute lung injuries. The lung tissue contains polyunsaturated fatty acids (PUFAs) that are substrates of lipid peroxidation that may lead to loss of the functional integrity of the cell membranes. In this study, we compare the in vitro protective effect of pulmonary surfactant protein A (SP-A), purified from porcine surfactant, against ascorbate-Fe(2+) lipid peroxidation stimulated by linoleic acid hydroperoxide (LHP) of the mitochondria and microsomes isolated from rat lung; deprived organelles of ascorbate and LHP were utilized as control. The process was measured simultaneously by chemiluminescence as well as by PUFA degradation of the total lipids isolated from these organelles. The addition of LHP to rat lung mitochondria or microsomes produces a marked increase in light emission; the highest value of activation was produced in microsomes (total chemiluminescence: 20.015+/-1.735 x 10(5) cpm). The inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (2.5 to 5.0 microg) of SP-A in rat lung mitochondria and 2.5 to 7.5 microg of SP-A in rat lung microsomes. The inhibitory effect reaches the highest values in the mitochondria, thus, 5.0 microg of SP-A produces a 100% inhibition in this membranes whereas 7.5 microg of SP-A produces a 51.25+/-3.48% inhibition in microsomes. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized membranes was found in the arachidonic acid content; this decreased from 9.68+/-1.60% in the native group to 5.72+/-1.64% in peroxidized mitochondria and from 7.39+/-1.14% to 3.21+/-0.77% in microsomes. These changes were less pronounced in SP-A treated membranes; as an example, in the presence of 5.0 microg of SP-A, we observed a total protection of 20:4 n-6 (9.41+/-3.29%) in mitochondria, whereas 7.5 microg of SP-A produced a 65% protection in microsomes (5.95+/-0.73%). Under these experimental conditions, SP-A produces a smaller inhibitory effect in microsomes than in mitochondria. Additional studies of lipid peroxidation of rat lung mitochondria or microsomes using equal amounts of albumin and even higher compared to SPA were carried out. Our results indicate that under our experimental conditions, BSA was unable to inhibit lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria or microsomes, thus indicating that this effect is specific to SP-A.     

The different cleavage DNA sequence specificity explains the camptothecin resistance of the human topoisomerase I Glu418Lys mutant

Yeast cells expressing the Glu418Lys human topoisomerase I mutant display a camptothecin resistance that slowly decreases as a function of time. Molecular characterization of the single steps of the catalytic cycle of the purified mutant indicates that it has a relaxation activity identical to the wild-type protein but a different DNA sequence specificity for the cleavage sites when compared to the wild-type enzyme, as assayed on several substrates. In particular the mutant has a low specificity for CPT sensitive cleavable sites. In fact, the mutant has, at variance of the wild-type enzyme, a reduced preference for cleavage sites having a thymine base in position −1 of the scissile strand. This preference, together with the strict requirement for a thymine base in position −1 for an efficient camptothecin binding, explains the temporary camptothecin resistance of the yeast cell expressing the mutant and points out the importance of the DNA sequence in the binding of the camptothecin drug.     

Evaluation of the MicroFoss system for the analysis of Escherichia coli in water

In this study, the performance of the MicroFoss system (Foss, Spain) for the enumeration of Escherichia coli in water samples was evaluated. One hundred and eighty-five samples were analysed both by MicroFoss assay and culture isolation on Tryptone-Bile X-glucuronide agar (TBX), and the correlation coefficient obtained was 0.92. The analysis of 28 new samples using both methods showed a statistically significant relationship at the 99.5% confidence level between log colony forming units obtained by MicroFoss assay and those obtained using growth on TBX agar. Nevertheless, when the level of sample contamination was low, the variability was high. In conclusion, the MicroFoss system is a rapid and simple alternative method for the enumeration of E. coli in water although discordance between the results using these methods in samples with low counts could limit its use for the study of clean water such as potable water.     

Akt activity protects rheumatoid synovial fibroblasts from Fas-induced apoptosis by inhibition of Bid cleavage

Introduction  

Synovial hyperplasia is a main feature of rheumatoid arthritis pathology that leads to cartilage and bone damage in the inflamed joints. Impaired apoptosis of resident synoviocytes is pivotal in this process. Apoptosis resistance seems to involve defects in the extrinsic and intrinsic apoptotic pathways. The aim of this study was to investigate the association of PI3Kinase/Akt and the mitochondrial apoptotic pathway in the resistance of rheumatoid arthritis (RA) fibroblast like synovial cells (FLS) to Fas-mediated apoptosis.     

Olive flowering trends in a large Mediterranean area (Italy and Spain)

The aim of this study was to investigate the main climatic and biological trends related to olive flowering in central-southern Italy compared to those in Andalusia, Spain. Results since 1982 were compared for the two long-series monitoring areas of Cordoba and Perugia, and since 1992–1999 for the short-series areas. The relationship between climatic trends and the biological response of the olive, a widespread culture in the Mediterranean basin, were investigated. An aerobiological method involving capturing pollen released into the atmosphere was utilised as a bioindicator of flowering phenology. The study results confirm the strong relationship between flowering periods and spring temperature trends for the olive. Temperature during March, April and May was the parameter most related to flowering date in the study areas, particularly in Italy. In some cases we found a significant correlation between flowering and past autumn temperatures, probably due to their effect on floral bud dormancy induction, but this phenomenon appeared to be of minor importance in the studied areas. The phenological trend results show the continuous advance of flowering dates to the late 1990s, followed by a relatively stationary time series related to a short-term temperature fluctuation in the Mediterranean area. This latter period probably represents a mesoscale event forced by a macroscale event—the North Atlantic Oscillation. The results reveal that the trend towards increased temperatures, and the consequent flowering advance of some species, indicated some years ago is nowadays not as clear as was expected and should be confirmed over the next few years in the Mediterranean areas under investigation.