The role of hydrogen bonding at the active site of a cupredoxin: the Phe114Pro azurin variant

The Phe114Pro mutation to the cupredoxin azurin (AZ) leads to a number of structural changes at the active site attributed to deletion of one of the hydrogen bonds to the Cys112 ligand, removal of the bulky phenyl group from the hydrophobic patch of the protein, and steric interactions made by the introduced Pro. The remaining hydrogen bond between the coordinating thiolate and the backbone amide of Asn47 is strengthened. At the type-1 copper site, the Cu(II)-O(Gly45) axial interaction decreases, while the metal moves out of the plane formed by the equatorial His46, Cys112, and His117 ligands, shortening the bond to the axially coordinating Met121. The resulting distorted tetrahedral geometry is distinct from the trigonal bipyramidal arrangement in the wild-type (WT) protein. The unique position of the main S(Cys) --> Cu(II) ligand-to-metal charge-transfer transition in AZ (628 nm) has shifted in the Phe114Pro variant to a value that is more typical for cupredoxins (599 nm). This probably occurs because of the removal of the Phe114-Cys112 hydrogen bond. The Phe114Pro mutation results in a 90 mV decrease in the reduction potential of AZ, and removal of the second hydrogen bond to the Cys ligand seems to be the major cause of this change. The C-terminal His117 ligand does not protonate in the reduced Phe114Pro AZ variant, which suggests that none of the structural features altered by the mutation are responsible for the absence of this effect in the WT protein. Upon reduction, the copper displaces further from the equatorial ligand plane and the Cu-S(Met121) bond length decreases. These changes are larger than those seen in the WT protein and contribute to the order of magnitude decrease in the intrinsic electron-transfer capabilities of the Phe114Pro variant.     

Shiga toxin facilitates its retrograde transport by modifying microtubule dynamics

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.     

Monotonicity of interleukin-1 receptor-ligand binding with respect to antagonist in the presence of decoy receptor

We consider the interaction between interleukin-1 IL-1, its receptor IL-1RI, the receptor antagonist IL-1Ra and a decoy receptor (or trap) that binds both with the ligand and the antagonist. We study how the interaction between IL-1Ra and the decoy receptor influences the effect of either reagent on reducing the equilibrium concentration of the receptor-ligand complex. We obtain that, given a certain relationship among the equilibrium constants and the total concentrations of solutes, IL-1Ra can reverse the effect of the decoy receptor of decreasing the equilibrium concentration of the receptor-ligand complex. This finding derives from a mathematical result applicable to any reversible chemical reaction system comprising four species arranged in a square such that each species binds its two immediate neighbors. The result gives the monotonicity of the equilibrium concentrations of the complex species as functions of the total concentrations of the simple species.     

Spatio-temporal distribution patterns and conservation of fish assemblages in a Chilean coastal river

River environments are characterized by extreme spatial and temporal variation in the physical environment. The relationship of fish assemblages to environmental variation is poorly understood in many systems. In Chile zonation patterns of fish assemblages have been documented in several Andean river drainages. Coastal river drainages are comparatively small, but inordinately important because of their highly endemic flora and fauna and their proximity to major human populations. For conservation purposes it is important to understand what environmental factors affect assemblage structure of fishes especially in the comparatively high diversity coastal drainages. We studied patterns of fish distribution and abundance in three rivers of the coastal, Andalien drainage near Concepción, Chile. We used multi-dimensional scaling analyses to compare patterns among zones (rithron, transition and potamon) and high and low flow seasons. Species assemblages differed by zone, but not with season. Assemblages consisted of nested subsets of species characterized by their range of distribution among zones. One species group was composed of widespread species that occurred in all three zones, another species group consisted of species found only in transitional and potamal zones, and a final group was comprised of species found only in the potamal zone. The potamal zone contained the most diverse and abundant fish assemblage. Fish assemblages were related to both water quality and habitat structure variables. This study suggests that the key to conserving the diversity of native fish communities in coastal Chilean rivers is in the conservation of potamal regions. Unfortunately, most protected areas in Chile are in the depauperate headwaters of drainages. Protection of only headwaters is clearly inadequate and will not contribute to the conservation of this unique freshwater fish fauna.     

Dendritic cell-derived IL-12p40 homodimer contributes to susceptibility in cutaneous leishmaniasis in BALB/c mice

Protection against Leishmania major in resistant C57BL/6 mice is mediated by Th1 cells, whereas susceptibility in BALB/c mice is the result of Th2 development. IL-12 release by L. major-infected dendritic cells (DC) is critically involved in differentiation of Th1 cells. Previously, we reported that strain differences in the production of DC-derived factors, e.g., IL-1alphabeta, are in part responsible for disparate disease outcome. In the present study, we analyzed the release of IL-12 from DC in more detail. Stimulated DC from C57BL/6 and BALB/c mice released comparable amounts of IL-12p40 and p70. In the absence of IL-4, BALB/c DC produced significantly more IL-12p40 than C57BL/6 DC. Detailed analyses by Western blot and ELISA revealed that one-tenth of IL-12p40 detected in DC supernatants was released as the IL-12 antagonist IL-12p40 homodimer (IL-12p80). BALB/c DC released approximately 2-fold more IL-12p80 than C57BL/6 DC both in vitro and in vivo. Local injection of IL-12p80 during the first 3 days after infection resulted in increased lesion volumes for several weeks in both L. major-infected BALB/c or C57BL/6 mice, in higher lesional parasite burdens, and decreased Th1-cytokine production. Finally, IL-12p40-transgenic C57BL/6 mice characterized by overexpression of p40 showed increased levels of serum IL-12p80 and enhanced disease susceptibility. Thus, in addition to IL-1alphabeta, strain-dependent differences in the release of other DC-derived factors such as IL-12p80 may influence genetically determined disease outcome.     

Overexpression of HBK3, a class I KNOX homeobox gene, improves the development of Norway spruce (Picea abies) somatic embryos

In order to investigate the effects of HBK3, a spruce gene member of the class I KNOX family, during somatic embryogenesis, sense (HBK3-S) and antisense (HBK3-A) Norway spruce (Picea abies) lines were generated. Somatic embryos produced from these lines were then analysed at morphological and structural levels. Compared with control, differentiation of immature somatic embryos from pro-embryogenic masses (PEMs) was accelerated in lines overexpressing HBK3 (HBK3-S). Such immature embryos showed enlarged embryogenic heads and were able to produce fully developed cotyledonary embryos at higher frequency. Furthermore, HBK3-S embryos had enlarged shoot apical meristems (SAMs) and enlarged expression pattern of PgAGO, a molecular marker gene specific to meristematic cells. Lines in which HBK3 (HBK3-A) was down-regulated had reduced ability to produce immature somatic embryos from PEMs and were not able to complete the maturation processes. To assess the function of HBK3 in comparison with that of angiosperm KNOX genes, this gene was ectopically expressed in Arabidopsis plants. As observed for spruce, Arabidopsis embryos overexpressing HBK3 had enlarged meristems and enlarged expression pattern of SHOOTMERISTEMLESS, a SAM molecular marker gene. In addition, transformed embryos were able to germinate at a higher rate and the resulting plants showed a variety of phenotypic aberrations, including abnormal leaves and reduced apical dominance. Overall, these data confirm the importance of KNOTTED genes during development and reveal the participation of HBK3 in conifer embryogeny. Furthermore, the results show redundant functions of this gene during embryonic growth of spruce and Arabidopsis, but not during post-embryonic growth.     

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.