Investigations of Sso7d catalytic residues by NMR titration shifts and electrostatic calculations

Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.     

In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.     

Multiple effects of paclitaxel are modulated by a high c-myc amplification level

Paclitaxel affects microtubule stability by binding to beta-tubulin, thus leading to cell accumulation in the G(2)/M phase, polyploidization, and apoptosis. Because both cell proliferation and apoptosis could be somehow regulated by the protooncogene c-myc, in this work we have investigated whether the c-myc amplification level could modulate the multiple effects of paclitaxel. To this aim, paclitaxel was administered to SW613-12A1 and -B3 human colon carcinoma cell lines (which are characterized by a high and low c-myc endogenous amplification level, respectively), and to the B3mycC5 cell line, with an enforced exogenous expression of c-myc copies. In this experimental system, we previously demonstrated that a high endogenous/exogenous level of amplification of c-myc enhances serum deprivation- and DNA damage-induced apoptosis. Accordingly, the present results indicate that a high c-myc amplification level potentiates paclitaxel cytotoxicity, confers a multinucleated phenotype, and promotes apoptosis to a great extent, thus suggesting that c-myc expression level is relevant in modulating the cellular responses to paclitaxel. We have recently shown in HeLa cells that the phosphorylated form of c-Myc accumulates in the nucleus, as distinct nucleolar and extranucleolar spots; here, we demonstrated that, after the treatment with paclitaxel, phosphorylated c-Myc undergoes redistribution, becoming diffused in the nucleoplasm.     

RecFOR function is required for DNA repair and recombination in a RecA loading-deficient recB mutant of Escherichia coli

The RecA loading activity of the RecBCD enzyme, together with its helicase and 5' --> 3' exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF(-) background the recB1080 mutant is recombination deficient, whereas in a recF(+) genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D(-)) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5' --> 3' exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.     

Micronucleus induction after whole-body microwave irradiation of rats

Adult male Wistar rats were exposed for 2 h a day, 7 days a week for up to 30 days to continuous 2,450 MHz radiofrequency microwave (rf/MW) radiation at a power density of 5-10 mW/cm(2). Sham-exposed rats were used as controls. After ether anesthesia, experimental animals were euthanized on the final irradiation day for each treated group. Peripheral blood smears were examined for the extent of genotoxicity, as indicated by the presence of micronuclei in polychromatic erythrocytes (PCEs). The results for the time-course of PCEs indicated significant differences (P<0.05) for the 2nd, the 8th and the 15th day between control and treated subgroups of animals. Increased influx of immature erythrocytes into the peripheral circulation at the beginning of the experiment revealed that the proliferation and maturation of nucleated erythropoietic cells were affected by exposure to the 2,450 MHz radiofrequency radiation. Such findings are indicators of radiation effects on bone-marrow erythropoiesis and their subsequent effects in circulating red cells. The incidence of micronuclei/1,000 PCEs in peripheral blood was significantly increased (P<0.05) in the subgroup exposed to rf/MW radiation after eight irradiation treatments of 2 h each in comparison with the sham-exposed control group. It is likely that an adaptive mechanism, both in erythrocytopoiesis and genotoxicity appeared in the rat experimental model during the subchronic irradiation treatment.     

Absence of endogenous interleukin-10 enhances the evolution of acute lung injury

Interleukin-10 (IL-10) exerts a wide spectrum of regulatory activities in the immune and inflammatory response. The aim of this study was to investigate the role of endogenous IL-10 on the modulation of the inflammatory response in mice subjected to carrageenan-induced lung injury. When compared to carrageenan-treated IL-10 wild-type (WT) mice, carrageenan-treated IL-10 knock-out mice (IL-10KO) mice experienced a higher rate of pleural exudation, and polymorphonuclear cell migration. Exudate levels of the pro-inflammatory cytokines tumour necrosis factor, interleukin-1beta and interleukin-6 were also greatly enhanced in IL-10KO mice in comparison to wild-type mice. Lung myeloperoxidase (MPO) activity was significantly reduced in IL-10WT mice when compared to IL-10KO mice-treated with carrageenan. The degree of oxidative and nitrosative damage was significantly higher in IL-10KO mice than in wild-type littermates, as indicated by elevated malondialdehyde levels and formation of nitrotyrosine and poly (ADP-ribose) synthetase (PARS). Staining of lung tissue sections obtained from carrageenan-treated IL-10WT with an anti-COX-2 antibody showed a positive staining of the inflamed tissue. Furthermore, expression of inducible nitric oxide synthase (iNOS) was found mainly in the macrophages of the inflamed lungs from carrageenan-treated IL-10WT mice. The intensity and degree of the staining for COX-2 and iNOS were markedly enhanced in tissue sections obtained from carrageenan-treated IL-10KO mice. Most notably, the degree of lung injury caused by carrageenan was also enhanced in IL-10KO mice. Taken together, our results clearly demonstrate that endogenous IL-10 exerts an anti-inflammatory role during acute inflammation and tissue damage associated with carrageenan-induced pleurisy, possibly by regulating neutrophil recruitment, and the subsequent cytokine and oxidant generation.     

Glacial history of the alpine herb Rumex Nivalis (Polygonaceae): a comparison of common phylogeographic methods with nested clade analysis

The glacial history of the alpine herb Rumex nivalis was investigated using amplified fragment length polymorphisms (AFLPs) and restriction fragment length polymorphisms with polymerase chain reaction (PCR-RFLPs) of cpDNA. Both traditional statistical methods widely applied in phylogeographic research and nested clade analysis were used. The AFLPs indicated little geographic structure probably due to the wind-pollinated reproductive system of the dioecious R. nivalis. Because cpDNA haplotypes exhibited distinct distributional patterns, correlation between AFLPs and PCR-RFLPs was low. The results of common statistical methods and of nested clade analysis were largely congruent. Both supported in situ survival of one group of common haplotypes in the Central Alps. For another group of common haplotypes, classical phylogeographic analyses gave strong evidence for survival in peripheral refugia at the northern alpine border, whereas this conclusion was not as clearly supported in the nested clade analysis. Nested clade analysis provided several detailed insights on past and ongoing populational demographic processes. Thus, it is a valuable tool in the phylogeographical analysis of haplotype data, but it should preferably be combined with other statistical analyses. In situations with low genetic variation in cpDNA, classical phylogeographic analytical tools on nuclear DNA will still be the methods of choice.