Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers

AimsUnder normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers.Main methodsCaco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin.Key findingsCytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix + PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization.SignificanceTreatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.     

Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficient mice

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.     

Rhizosphere persistence of three Pythium oligandrum strains in tomato soilless culture assessed by DNA macroarray and real-time PCR

In tomato soilless culture, plant-disease optimal control and growth promotion are achieved when the rhizosphere is heavily colonized by the biocontrol agent Pythium oligandrum. Discrepancies in performance are generally attributed to the poor persistence of P. oligandrum on roots. In this study, three selected strains of P. oligandrum were introduced into the rhizosphere of greenhouse-grown tomato plants, and their persistence was assessed by DNA macroarray hybridization and real-time PCR. The experimental data from DNA detection and plate counting were compared. PCR-based methods detected P. oligandrum throughout the 6-month growing season, whereas plate counting indicated its presence only over the first 3 months. Moreover, the DNA array method provided information about the various Pythium species present in the rhizosphere: P. dissotocum was frequently detected on roots of plants, without distinction between plants inoculated or not inoculated with the antagonist. The detection of other Pythium species was noticed sporadically (P. ultimum, P. sylvaticum and P. intermedium), independent of the treatment. Even though the yield enhancement is not significant throughout the entire growing season, data obtained from epidemiological studies demonstrate an enhancement of P. oligandrum persistence on the rhizosphere of plants and less use of mycoparasitism.     

Are Primates Ecosystem Engineers?

Animals can play important roles in structuring the plant communities in which they live. Some species are particularly influential in that they modify the physical environment by changing, maintaining, and/or creating new habitats; the term ecosystem engineer has been used to describe such species. We here assess the two major foraging strategies of primates, frugivory and folivory, in terms of the potential for primates to function as ecosystem engineers. We argue that whereas the role of primates as seed dispersers has received a great deal of attention, the potential role that folivorous primates play in structuring their environment through herbivory has received much less attention. Further, while quantifying if frugivorous primates are ecosystem engineers through their seed dispersal has proved very difficult, it is not as difficult to ascertain whether folivorous primates are ecosystem engineers. We document situations in which folivorous primates act as ecosystem engineers by 1) eating the leaves and/or bark of trees to the extent that they kill trees, 2) feeding on trees to the degree that they slow their growth relative to nonpreferred tree species, 3) eating the flowers of species to the extent that it does not set fruit, or 4) feeding on plants in such a way as to increase their productivity and abundance. Because evidence from the literature is very limited, where possible we present new evidence of these processes from the colobus monkeys at our long-term field site in Kibale National Park, Uganda. We conclude by discussing promising research programs that could be established to refine our understanding of the role primates play in shaping the structure of plant communities, especially tropical forests.     

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.     

Effect of chronic hyperoxic exposure on duroquinone reduction in adult rat lungs

NAD(P)H:quinone oxidoreductase 1 (NQO1) plays a dominant role in the reduction of the quinone compound 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ) to durohydroquinone (DQH2) on passage through the rat lung. Exposure of adult rats to 85% O2 for > or =7 days stimulates adaptation to the otherwise lethal effects of >95% O2. The objective of this study was to examine whether exposure of adult rats to hyperoxia affected lung NQO1 activity as measured by the rate of DQ reduction on passage through the lung. We measured DQH2 appearance in the venous effluent during DQ infusion at different concentrations into the pulmonary artery of isolated perfused lungs from rats exposed to room air or to 85% O2. We also evaluated the effect of hyperoxia on vascular transit time distribution and measured NQO1 activity and protein in lung homogenate. The results demonstrate that exposure to 85% O2 for 21 days increases lung capacity to reduce DQ to DQH2 and that NQO1 is the dominant DQ reductase in normoxic and hyperoxic lungs. Kinetic analysis revealed that 21-day hyperoxia exposure increased the maximum rate of pulmonary DQ reduction, Vmax, and the apparent Michaelis-Menten constant for DQ reduction, Kma. The increase in Vmax suggests a hyperoxia-induced increase in NQO1 activity of lung cells accessible to DQ from the vascular region, consistent qualitatively but not quantitatively with an increase in lung homogenate NQO1 activity in 21-day hyperoxic lungs. The increase in Kma could be accounted for by approximately 40% increase in vascular transit time heterogeneity in 21-day hyperoxic lungs.     

Life-history correlates of steroid concentrations in wild peripartum baboons

Steroid concentrations during late pregnancy and early lactation may be affected by both a female's reproductive history and her current condition, and may in turn predict subsequent life-history events, such as offspring survival. This study investigated these relationships in a wild primate population through the use of fecal steroid analysis in repeated sampling of peripartum baboons (Papio cynocephalus). Fecal samples were collected from 32 females in five groups within the Amboseli basin during 8 weeks prior to parturition and 13 weeks postpartum. From December 1999 through February 2002, 176 fecal samples were collected from individuals representing 39 peripartum periods. Fecal concentrations of progestins (fP), estrogen metabolites (fE), glucocorticoids (fGC), and testosterone metabolites (fT) were measured by radioimmunoassay. Steroid concentrations declined from late pregnancy to lactation, and the decline was greatest and most precipitous for fE and fP. Primiparous females had significantly higher mean fE concentrations in each of the last 2 months of pregnancy compared to multiparous females. Among multiparous females, fE and fT were significantly higher during late pregnancy in females carrying a male fetus compared to those carrying a female fetus. During early lactation, high fT in young mothers predicted subsequent infant death during the first year of life. These findings illustrate the potential power of repeated fecal-steroid sampling to elucidate mechanisms of life-history variability in natural populations. They also document significant differences in hormone profiles among subgroups, and highlight that such normative subgroup information is essential for interpreting individual variability in hormone-behavior associations.     

Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes

Pathway specificity is poorly understood for mitogen-activated protein kinase (MAPK) cascades that control different outputs in response to different stimuli. In yeast, it is not known how the same MAPK cascade activates Kss1 MAPK to promote invasive growth (IG) and proliferation, and both Fus3 and Kss1 MAPKs to promote mating. Previous work has suggested that the Kss1 MAPK cascade is activated independently of the mating G protein (Ste4)-scaffold (Ste5) system during IG. Here we demonstrate that Ste4 and Ste5 activate Kss1 during IG and in response to multiple stimuli including butanol. Ste5 activates Kss1 by generating a pool of active MAPKKK (Ste11), whereas additional scaffolding is needed to activate Fus3. Scaffold-independent activation of Kss1 can occur at multiple steps in the pathway, whereas Fus3 is strictly dependent on the scaffold. Pathway specificity is linked to Kss1 immunity to a MAPK phosphatase that constitutively inhibits basal activation of Fus3 and blocks activation of the mating pathway. These findings reveal the versatility of scaffolds and how a single MAPK cascade mediates different outputs.     

Actin dynamics in plant cells: a team effort from multiple proteins orchestrates this very fast-paced game

Gazing at a giant redwood tree in the Pacific Northwest, that has grown to enormous heights over centuries, does little to convince one that plants are built for speed and versatility. Even at the cellular level, a system of polymers-the cell skeleton or cytoskeleton-integrates signals and generates subcellular structures spanning scales of a few nanometers to hundreds of micrometers that coordinate cell growth. The term cytoskeleton itself connotes a stable structure. Clearly, this is not the case. Recent studies using advanced imaging modalities reveal the plant actin cytoskeleton to be a highly dynamic, ever changing assemblage of polymers. These insights along with growing evidence about the biochemical/biophysical properties of plant cytoskeletal polymers, especially those obtained by single filament imaging and reconstituted systems of purified proteins analyzed by total internal reflection fluorescence microscopy, allow the generation of a unique model for the dynamic turnover of actin filaments, termed stochastic dynamics. Here, we review several significant advances and highlight opportunities that will position plants at the vanguard of research on actin organization and turnover. A challenge for the future will be to apply the power of reverse-genetics in several model organisms to test the molecular details of this new model.