Relationship between Riparian Vegetation and Stream Benthic Communities at Three Spatial Scales

We examined the influence of riparian vegetation on macroinvertebrate community structure in streams of the Upper Thames River watershed in southwestern Ontario. Thirty-three μ-basins (129–1458 ha) were used to identify land cover variables that influenced stream macroinvertebrates. Micro-basins represented the entire drainage area of study streams and were similar in stream order (first, second) and land cover (agricultural or forest; no urban). We described the structure and composition of riparian vegetation and benthic macroinvertebrate communities at the outflow reach. The nature of the land cover was quantified for the stream network buffer (30 m) and the whole μ-basin. The objective of this study was to measure the magnitude and nature of the relationship between the riparian vegetation and benthic macroinvertebrate community at the outflow reach, stream network buffer, and whole μ-basin scales. Taxon richness (including total number of Ephemeroptera, Plecoptera, and Trichoptera taxa) and Simpson’s diversity of the macroinvertebrate community all increased with increased tree cover in the riparian zone at the outflow reach scale. Simpson’s equitability was lower with greater agricultural land cover in the stream network buffer. No relationship between the macroinvertebrate community and land cover was found at the whole μ-basin scale. Analysis of the influence of land cover on stream communities within a spatial hierarchy is important for understanding the interactions of stream ecosystems with their adjacent landscapes.     

Organismal differences in post-translational modifications in histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.     

Fragile X related protein 1 isoforms differentially modulate the affinity of fragile X mental retardation protein for G-quartet RNA structure

Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of expression of the Fragile X Mental Retardation Protein (FMRP), an RNA binding protein with high specificity for G-quartet RNA structure. FMRP is involved in several steps of mRNA metabolism: nucleocytoplasmic trafficking, translational control and transport along dendrites in neurons. Fragile X Related Protein 1 (FXR1P), a homologue and interactor of FMRP, has been postulated to have a function similar to FMRP, leading to the hypothesis that it can compensate for the absence of FMRP in Fragile X patients. Here we analyze the ability of three isoforms of FXR1P, expressed in different tissues, to bind G-quartet RNA structure specifically. Only the longest FXR1P isoform was found to be able to bind specifically the G-quartet RNA, albeit with a lower affinity as compared to FMRP, whereas the other two isoforms negatively regulate the affinity of FMRP for G-quartet RNA. This result is important to decipher the molecular basis of fragile X syndrome, through the understanding of FMRP action in the context of its multimolecular complex in different tissues. In addition, we show that the action of FXR1P is synergistic rather than compensatory for FMRP function.     

Cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants: saponin, incomplete Freund's adjuvant, and monophosphoryl lipid A

Vaccine adjuvants are substances associated with antigens that are fundamental to the formation of an intense, durable, and fast immune response. In this context, the use of vaccine adjuvants to generate an effective cellular immune response is crucial for the design and development of vaccines against visceral leishmaniasis. The objective of this study was to evaluate innate inflammatory response induced by the vaccine adjuvants saponin (SAP), incomplete Freund's adjuvant (IFA), and monophosphoryl lipid A (MPL). After a single dose of adjuvant was injected into the skin of mice, we analyzed inflammatory reaction, selective cell migration, and cytokine production at the injection site, and inflammatory cell influx in the peripheral blood. We found that all vaccine adjuvants were able to promote cell recruitment to the site without tissue damage. In addition, they induced selective migration of neutrophils, macrophages, and lymphocytes. The influx of neutrophils was notable at 12 h in all groups, but at other time points it was most evident after inoculation with SAP. With regard to cytokines, the SAP led to production of interleukin (IL)-2, IL-6, and IL-4. IFA promoted production of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, IL-17, IL-4, and IL-10. We also observed that MPL induced high production of IL-2, TNF-α, and IFN-γ, in addition to IL-6, IL-17, and IL-10. In peripheral blood, values of certain cell populations in the local response changed after stimulation. Our data demonstrate that the three vaccine adjuvants stimulate the early events of innate immune response at the injection site, suggesting their ability to increase the immunogenicity of co-administered antigens. Moreover, this work provides relevant information about elements of innate and acquired immune response induced by vaccine adjuvants administered alone.     

Preeclampsia: current understanding of the molecular basis of vascular dysfunction

Preeclampsia is a pregnancy-specific disorder characterised by hypertension and proteinuria occurring after the 20th week of gestation. Delivery of the placenta results in resolution of the condition, implicating the placenta as a central culprit in the pathogenesis of preeclampsia. In preeclampsia, an inadequate placental trophoblast invasion of the maternal uterine spiral arteries results in poor placental perfusion, leading to placental ischaemia. This could result in release of factors into the maternal circulation that cause widespread activation or dysfunction of the maternal endothelium. Factors in the maternal circulation might induce oxidative stress and/or elicit an inflammatory response in the maternal endothelium, resulting in the altered expression of several genes involved in the regulation of vascular tone. This review addresses the potential circulating factors and the molecular mechanisms involved in the alteration of vascular function that occurs in preeclampsia.     

Heterozygote advantage at MHC DRB may influence response to infectious disease epizootics

The effect of MHC polymorphism on individual fitness variation in the wild remains equivocal; however, much evidence suggests that heterozygote advantage is a major determinant. To understand the contribution of MHC polymorphism to individual disease resistance or susceptibility in natural populations, we investigated two MHC class II B loci, DQB and DRB, in the New Zealand sea lion (NZSL, Phocarctos hookeri). The NZSL is a threatened species which is unusually susceptible to death by bacterial infection at an early age; it has suffered three bacterial induced epizootics resulting in high mortality levels of young pups since 1997. The MHC DQB and DRB haplotypes of dead NZSL pups with known cause of death (bacteria, enteritis or trauma) were sequenced and reconstructed, compared to pups that survived beyond 2 months of age, and distinct MHC DRB allele frequency and genotype differences were identified. Two findings were striking: (i) one DRB allele was present only in dead pups, and (ii) one heterozygous DRB genotype, common in live pups, was absent from dead pups. These results are consistent with some functional relationship with these variants and suggest heterozygote advantage is operating at DRB. We found no association between heterozygosity and fitness at 17 microsatellite loci, indicating that general heterozygosity is not responsible for the effect on fitness detected here. This result may be a consequence of recurrent selection by multiple pathogen assault over recent years and highlights the importance of heterozygote advantage at MHC as a potential mechanism for fitness differences in wild populations.     

Endogenous VEGF-A is responsible for mitogenic effects of MCP-1 on vascular smooth muscle cells

Vessel wall remodeling is a complex phenomenon in which the loss of differentiation of vascular smooth muscle cells (VSMCs) occurs. We investigated the role of rat macrophage chemoattractant protein (MCP)-1 on rat VSMC proliferation and migration to identify the mechanism(s) involved in this kind of activity. Exposure to very low concentrations (1-100 pg/ml) of rat MCP-1 induced a significant proliferation of cultured rat VSMCs assessed as cell duplication by the counting of total cells after exposure to test substances. MCP-1 stimulated VSMC proliferation and migration in a two-dimensional lateral sheet migration of adherent cells in culture. Endogenous vascular endothelial growth factor-A (VEGF-A) was responsible for the mitogenic activity of MCP-1, because neutralizing anti-VEGF-A antibody inhibited cell proliferation in response to MCP-1. On the contrary, neutralizing anti-fibroblast growth factor-2 and anti-platelet-derived growth factor-bb antibodies did not affect VSMC proliferation induced by MCP-1. RT-PCR and Western blot analyses showed an increased expression of either mRNA or VEGF-A protein after MCP-1 activation (10-100 pg/ml), whereas no fms-like tyrosine kinase (Flt)-1 receptor upregulation was observed. Because we have previously demonstrated that hypoxia (3% O2) can enhance VSMC proliferation induced by VEGF-A through Flt-1 receptor upregulation, the effects of hypoxia on the response of VSMCs to MCP-1 were investigated. Severe hypoxia (3% O2) potentiated the growth-promoting effect of MCP-1, which was able to significantly induce cell proliferation even at a concentration as low as 0.1 pg/ml. These findings demonstrate that low concentrations of rat MCP-1 can directly promote rat VSMC proliferation and migration through the autocrine production of VEGF-A.     

Hydrogen-oxidizing capabilities of Helicobacter hepaticus and in vivo availability of the substrate

Hydrogen-oxidizing hydrogenase activity was detected in Helicobacter hepaticus and compared to the activity in Helicobacter pylori for characteristics associated with hydrogen uptake respiratory hydrogenases. Intact whole cells could couple H(2) oxidation to oxygen uptake, and no H(2) uptake was observed without oxygen available to complete the respiratory pathway. The H. hepaticus enzyme coupled H(2) oxidation to reduction of many positive potential acceptors, and it underwent anaerobic or reductive activation. H. hepaticus had a strong affinity for molecular H(2) (apparent K(m) of 2.5 micro M), and microelectrode measurements on the livers of live mice demonstrated that H(2) is available in the host tissue at levels 20-fold greater than the apparent whole-cell K(m) value.     

Translocation of a phycoerythrin alpha subunit across five biological membranes

Cryptophytes, unicellular algae, evolved by secondary endosymbiosis and contain plastids surrounded by four membranes. In contrast to cyanobacteria and red algae, their phycobiliproteins do not assemble into phycobilisomes and are located within the thylakoid lumen instead of the stroma. We identified two gene families encoding phycoerythrin alpha and light-harvesting complex proteins from an expressed sequence tag library of the cryptophyte Guillardia theta. The proteins bear a bipartite topogenic signal responsible for the transport of nuclear encoded proteins via the ER into the plastid. Analysis of the phycoerythrin alpha sequences revealed that more than half of them carry an additional, third topogenic signal comprising a twin arginine motif, which is indicative of Tat (twin arginine transport)-specific targeting signals. We performed import studies with several derivatives of one member using a diatom transformation system, as well as intact chloroplasts and thylakoid vesicles isolated from pea. We demonstrated the different targeting properties of each individual part of the tripartite leader and show that phycoerythrin alpha is transported across the thylakoid membrane into the thylakoid lumen and protease-protected. Furthermore, we showed that thylakoid transport of phycoerythrin alpha takes place by the Tat pathway even if the 36 amino acid long bipartite topogenic signal precedes the actual twin arginine signal. This is the first experimental evidence of a protein being targeted across five biological membranes.     

Human conjunctival epithelial precursor cells and their progeny in 3D organotypic culture

We report on an in vitro organ culture method to investigate human conjunctival epithelial basal precursor cells and their progeny within a more natural three-dimensional microenvironment. Conjunctival fragments were cultured on gelatin sponges in medium with 10% FBS. The conjunctival phenotype of the epithelium was confirmed by the expression and distribution of a panel of markers (p63, CK-13/CK-10, CK-19, Ki-67, PAS for goblet cells, CD45 for infiltrating interlamellar leukocytes and nestin for mesenchymal and ocular epithelial precursor cells). After 7 days, the epithelium had exfoliated its superficial layers (mostly CK-19( )positive cells and all goblets), maintaining only 1-2 layers of basal/parabasal cells, p63, CK-13/CK-10 and nestin positive cells, firmly attached to the specimen. After 14 days, a new multilayered epithelium was formed, consisting of p63, CK-13/CK-10, nestin positive cells and in the high-zone CK-19 positive cells with new goblets. Additionally, we found interlamellar leukocytes which had probably migrated from capillaries that continued to be well maintained in the subepithelial stroma. Cells dispersed from conjunctival epithelium and co-cultured with feeder post-mitotic NIH3T3 fibroblasts formed mosaics displaying a basal epithelial phenotype. These cells expressed CD133 as revealed by RT-PCR. These organ cultures provide new opportunities to investigate epithelial reconstitution of the conjunctival surface and changes that may have occurred to their stem/precursor cells during adaptation to varying conditions in vitro.