NiFe hydrogenase active site biosynthesis: identification of Hyp protein complexes in Ralstonia eutropha

Biosynthesis of the NiFe hydrogenase active site is a complex process involving the action of the Hyp proteins: HypA-HypF. Here we investigate the mechanism of NiFe site biosynthesis in Ralstonia eutropha by examining the interactions between HypC, HypD, HypE, and HypF1. Using an affinity purification procedure based on the Strep-tag II, we purified HypC and HypE from different genetic backgrounds as complexes with other hydrogenase-related proteins and characterized them using immunological analysis. Copurification of HypC and HoxH, the active site-containing subunit of the soluble hydrogenase in R. eutropha, from several different genetic backgrounds suggests that this complex forms early in the maturation process. With respect to the Hyp proteins, it is shown that HypE and HypF1 formed a stable complex both in vivo and in vitro. Furthermore, HypC and HypD functioned as a unit. Together, they were able to interact with HypE to form a range of complexes probably varying in stoichiometry. The HypC/HypD/HypE complexes did not involve HypF1 but appeared to be more stable when HypF1 was also present in the cells. We hypothesize that HypF1 is able to modify some component of the HypC/HypD/HypE complex. Since we have also seen that HypF1 and HypE form a complex, it is likely that HypF1 modifies HypE. On the basis of these results, we propose a complete catalytic cycle for HypE. First, it is modified by HypF1, and then it can form a complex with HypC/HypD. This activated HypE/HypC/HypD complex could then decompose by donating active site components to the immature hydrogenase and regenerate unmodified HypE.     

KIBRA is a novel substrate for protein kinase Czeta

WW domain-containing proteins are found in all eukaryotic cells and they are involved in the regulation of a wide variety of cellular functions. We recently identified the neuronal protein KIBRA as novel member of this family of signal transducers. In this report, we describe the identification of protein kinase C (PKC) zeta as a KIBRA-interacting protein. PKCzeta is known to play an important role in synaptic plasticity and memory formation but its specific targets are not well known. Our studies presented here revealed that KIBRA is a novel substrate for PKCzeta and suggest that PKCzeta phosphorylation may regulate the cellular function of KIBRA.     

Cytokine dysregulation induced by apoptotic cells is a shared characteristic of macrophages from nonobese diabetic and systemic lupus erythematosus-prone mice

Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1 beta and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1 beta by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.     

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.     

Neurogenesis in myriapods and chelicerates and its importance for understanding arthropod relationships

Several alternative hypotheses on the relationships betweenthe major arthropod groups are still being discussed. We reexaminehere the chelicerate/myriapod relationship by comparing previouslypublished morphological data on neurogenesis in the euarthropodgroups and presenting data on an additional myriapod (Strigamiamaritima). Although there are differences in the formation ofneural precursors, most euarthropod species analyzed generateabout 30 single neural precursors (insects/crustaceans) or precursorgroups (chelicerates/myriapods) per hemisegment that are arrangedin a regular pattern. The genetic network involved in recruitmentand specification of neural precursors seems to be conservedamong euarthropods. Furthermore, we show here that neural precursoridentity seems to be achieved in a similar way. Besides theseconserved features we found 2 characters that distinguish insects/crustaceansfrom myriapods/chelicerates. First, in insects and crustaceansthe neuroectoderm gives rise to epidermal and neural cells,whereas in chelicerates and myriapods the central area of theneuroectoderm exclusively generates neural cells. Second, neuralcells arise by stem-cell-like divisions of neuroblasts in insectsand crustaceans, whereas groups of mainly postmitotic neuralprecursors are recruited for the neural fate in cheliceratesand myriapods. We discuss whether these characteristics representa sympleisiomorphy of myriapods and chelicerates that has beenlost in the more derived Pancrustacea or whether these characteristicsare a synapomorphy of myriapods and chelicerates, providingthe first morphological support for the Myriochelata group.     

The fate of sheep-dispersed seeds: Plant species emergence and spatial patterns

Sheep epizoochory has often been proposed as an important vector which can help to overcome the dispersal limitation of plants in fragmented landscapes. In order to evaluate the contribution of herbivores to recruitment especially of target species, the dispersal and post-dispersal fate of such seeds must be known. In a field experiment sheep with seeds of mainly target species (experimentally attached to their coats) were present at three sand plots for 24 h. Natural epizoochorous dispersal was already shown for most of the species in our study area. Seed detachment, trampling intensity and seed shadow were measured; seedling emergence and survival were recorded over an 8-month period. In addition, the effect of sheep trampling on seedling emergence and survival of two threatened species, Jurinea cyanoides and Koeleria glauca, were studied.     

Occurrence, diversity and community structures of peracarid crustaceans (Crustacea, Malacostraca) along the southern shelf of Greenland

The species composition of peracarids (Crustacea: Malacostraca) of the Greenland shelf between 60°N and 65°N was investigated by means of 10 qualitative epibenthic samples in relation to environmental factors. In total, 59,234 specimens were collected belonging to 219 species. The relative abundance was much higher on the western shelf (total of 41,594 specimens) than on the eastern shelf (total of 17,640 specimens with same effort). Three species were new to science, while five records were new for the investigated area. The species composition was dominated by amphipods (58%), while the relative abundances of isopods (25%), cumaceans (11%) and tanaidaceans (6%) were much lower. Diversity and evenness were similar in the eastern and the western areas. Multivariate analyses of the species relative abundances divided the peracarids into a southeastern and southwestern Greenland fauna. Based on a correlation analysis between faunal data and five environmental variables, the separation between the two areas was mainly based on sediment type. Species contributing most to the separation between eastern and western fauna included the amphipods Hardametopa nasuta, Photis reinhardi and Phoxocephalus holboelli, the isopods Pleurogonium spinosissimum, Iolella laciniata and Nannoniscus oblongus and the cumaceans Leucon cf. nasicoides and Campylaspis horrida. Species distribution patterns are discussed in the light of habitat and feeding preferences.     

Recognition of Apoptotic Cells by Epithelial Cells: CONSERVED VERSUS TISSUE-SPECIFIC SIGNALING RESPONSES*

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (mϕ). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence mϕ survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like mϕ, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and mϕ. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and mϕ. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in mϕ, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from mϕ. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.     

Rationally Evolving MCP-1/CCL2 into a Decoy Protein with Potent Anti-inflammatory Activity in Vivo

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.     

Effect of Campoletis sonorensis ichnovirus cys-motif proteins on Heliothis virescens larval development

Polydnaviruses are obligate symbionts of some parasitic hymenopteran wasps responsible for modifying the physiology of their host lepidopteran larvae to benefit the endoparasite. Injection of Campoletis sonorensis ichnovirus (CsIV) into Heliothis virescens larvae alters larval growth, development and immunity but genes responsible for alterations of host physiology are not well described. Recent studies of polydnavirus genomes establish that these genomes encode families of related genes expressed in parasitized larvae. Here we evaluate five members of the CsIV cys-motif gene family for their ability to inhibit growth and development of lepidopteran larvae. To study the function of cys-motif proteins, recombinant proteins were produced from baculovirus expression vectors and injected or fed to H. virescens larvae in diet. rVHv1.1 was identified as the most potent protein tested causing a significant reduction in growth of H. virescens and Spodoptera exigua larvae. H. virescens larvae ingesting this protein also exhibited delayed development, reductions in pupation and increased mortality. Increased mortality was associated with chronic sub-lethal baculovirus infections. Taken together, these data indicate that the cys-motif proteins have pleiotropic effects on lepidopteran physiology affecting both development and immunity.