Different rates of nucleotide substitutions in Wolbachia endosymbionts of arthropods and nematodes: arms race or host shifts?

The genus Wolbachia encompasses intracellular bacteria found in arthropods and in filarial nematodes. In arthropods, Wolbachia is primarily a reproductive parasite and shows relatively frequent horizontal transfer between host species, while in nematodes it appears to be a mutualist and is strictly vertically transmitted. We can expect that different selective pressures are acting on their genomes. Here we present an analysis of three Wolbachia genes, wsp, ftsZ and dnaA. In wsp of arthropod Wolbachia, an excess of non-synonymous substitutions was observed, providing evidence for positive selection. In nematode Wolbachia, no evidence for positive selection was found. Pressure for amino acid variation in wsp of arthropod Wolbachia could derive either from an arms race with the host or from the occurrence of more frequent hosts shifts due to horizontal transmission. In nematode Wolbachia, the lack of positively selected sites could result from the absence of an arms race, or from the homogeneity of the biochemical environment they exist in (ensured by strict vertical transmission). In ftsZ minor differences in substitution patterns were observed between arthropod and nematode Wolbachia, only in the 3'-portion of the gene. dnaA showed comparable patterns of variation in both lineages, with evidence for strong conservation.

Improved method for the allelic definition of C4A and C4B polymorphism (HLA class III).

The polymorphism of the fourth component of human serum complement (C4) is well established at the proteinic level; at the DNA level in the analysis of C4A and C4B gene polymorphism, the PCR technique is not widely and routinely used because it is time consuming and still presents reproducibility problems. This is a serious problem because only PCR genotyping allows the establishment of Rodgers-Chido reverse antigenicity without the need for classical family segregation studies, whose samples are not always easy to obtain. The most commonly used protocol requires an initial PCR followed by nested amplification of all the products supposed positive or negative. The two reactions are set up using differing cycling conditions, primers, and magnesium chloride concentrations. We developed a simplified procedure to easily obtain reproducible results and used a single protocol for all reactions. Nested PCR is made using only the positive samples, so we decrease the number of samples to handle, the time spent for the work, and the reagents used for the reactions. Moreover, we increased the reproducibility of the experiments.

Characteristics of the response of ovine granulocytes (PMNs) to zymosan-activated serum (ZAS) and to recombinant human interleukin-8 (IL-8).

The chemotactic activity of zymosan-activated serum (ZAS) and of two concentrations of recombinant human IL-8 (IL-8(25), 25 ng/ml; IL-8(50), 50 ng/ml) for ovine polymorphonuclear granulocytes (PMNs) was tested in a modified Boyden chamber. Thick cellulose acetate filters and the leading front method were used to quantify the movements of the cells. Both ZAS and IL-8(25) exerted a chemotactic effect on ovine PMNs (P < 0.01): IL-8(50) induced a more homogeneous response (P < 0.001). To verify the characteristics of the responsiveness to the chemokines after short-term (st) or long-term (lt) repeated samplings, chemotaxis was investigated 1 (T1st), 2 (T2st), 24 (T3st) and 48 h (T4st) after the basal sampling (T0st) and 15 days (T1lt) after the basal sampling (T0lt). No differences in chemotaxis were found in long-term repeated samplings. In contrast an increase in the responsiveness to IL-8(25) and to IL-8(50) (P < 0.05) was detected at T2st in comparison with T0st. Furthermore, the significance of the distance run by activated PMNs compared with the controls, increased from T0st to T2st, as a sign of a more homogeneous response to the chemokines. In the absence of evident changes in circulating leucocyte numbers and in serum cortisol concentrations, these findings could be interpreted as a consequence of a different expression of chemoattractant receptors on the membrane of PMNs collected at different times.

Cytoskeleton actin changes in IL-2 activated cells.

In the present study we analysed the changes in cytoskeleton actin in lymphoid cells following IL-2 activation and during cell interactions by means of light and electron microscopy, immunofluorescence and molecular analysis. By morphological analysis we observed a higher fluorescence in the activated cells than in the quiescent ones with no modifications in the cytoskeleton pattern comparing activated to resting cells. The results of molecular analysis indicate that, after IL-2 activation, there is a reorganisation of the actin component of the cell cytoskeleton accompanied by the differential expression of the corresponding genes. A future study will be extended to the analysis of others components of the cytoskeleton network.

Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development.

p38alpha MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38alpha gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. Our results indicate that p38alpha is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.

Identification of mRNAs differentially expressed in lymphocytes following interleukin-2 activation.

We investigated genes involved in the interleukin-2 activation of cultured lymphocytes using a differential display reverse transcription PCR technique. Three cDNA fragments corresponding to mRNAs differentially amplified in the activated lymphocytes were sequenced and identified. These fragments were identical to the 3' region of the mRNAs encoding for the tumor rejection antigen TRA 1 that is the human homologue of the murine heat shock protein gp96, the DAP12 protein that possesses an immunoreceptor tyrosine-based activation motif, and the human motor protein p87/89 expressed in the heart. These proteins are involved, respectively, in cellular communication, in signal transduction, and in cellular movements. Our findings suggest that the activation of cellular immune response by interleukin-2 is a process analogous to other known phenomena of activation of catabolic reactions of energy transduction for activities which allow adaptation of cells to stress conditions.