A novel non-synonymous polymorphism (p.Arg240His) in C4b-binding protein is associated with atypical hemolytic uremic syndrome and leads to impaired alternative pathway cofactor activity

Atypical hemolytic uremic syndrome (aHUS) is a disorder characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations, polymorphisms, and copy number variation in complement factors and inhibitors are associated with aHUS. In this study, we report the first functional non-synonymous polymorphism in the complement inhibitor C4b-binding protein (C4BP) alpha-chain (c.719G>A; p.Arg240His), which is associated with aHUS. This heterozygous change was found in 6/166 aHUS patients compared with 5/542 normal (chi2 = 6.021; p = 0.014), which was replicated in a second cohort of aHUS patients in which we found 5/170 carriers. The polymorphism does not decrease expression efficiency of C4BP. p.Arg240His is equally efficient as the wild type in binding and supporting degradation of C4BP but its ability to bind C3b and act as cofactor to its degradation both in fluid phase and on surfaces is impaired. This observation supports the hypothesis that dysregulation of the alternative pathway of complement is pivotal for aHUS. Three of the patients carry also mutations in membrane cofactor protein and factor H strengthening the hypothesis that individuals may carry multiple susceptibility factors with an additive effect on the risk of developing aHUS.     

Altered Tumor Biology and Tumorigenesis in Irradiated and Chemical Carcinogen-Treated Single and Combined Connexin32/p27Kip1-Deficient Mice

Connexin32 knockout mice (Cx32-KO) exhibit increased chemical and radiation-induced liver and lung tumorigenesis. This increased tumor incidence is associated with altered tumor biology including enhanced tumor progression and an increased percent of MAPK-active tumors. Likewise, mice lacking the tumor suppressor/cell cycle regulator p27^Kip1 exhibit increased tumorigenesis in a variety of tissues following chemical and radiation induction. Interestingly, in a double-deficient mouse model (DKO), additional loss of p27^Kip1 in a Cx32-KO background results in attenuation of liver and lung tumorigenesis as well as MAPK activation profiles, suggesting pathway interaction. While these mouse strains exhibit altered liver and lung tumor susceptibility following both chemical (DEN) and radiation (X-ray) induction protocols, comparisons of the resulting tumor incidence, multiplicity, tumor progression, and MAPK activation in response to these two distinct carcinogens underscores the separate influence of each individual gene on both tumor formation and activation of specific oncogenic pathways. Furthermore, these studies demonstrate that different carcinogens interact disparately with Cx32/p27^Kip1 genotypic backgrounds in situ resulting in varied tumorigenic response.     

Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells

Cells expressingconnexin43 are able to upregulate gap junction (GJ) communication byenhancing the assembly of new GJs, apparently through increasedconnexin trafficking. Because G proteins are known to regulatedifferent aspects of protein trafficking, we examined the effects ofpertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJassembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicatedby a reduction in dye transfer. Electron microscopy also revealed a60% decrease in the number of GJ channels per cell interface.Importantly, PTX blocked the twofold enhancement in GJ assembly foundin the presence of low-density lipoprotein. Two G_iproteins (G_i2 and G_i3), which have beenimplicated in the control of membrane trafficking, reacted with PTX inADP-ribosylation studies. PTX and/or the trafficking inhibitors,brefeldin A and monensin, inhibited GJ assembly to comparable degrees.In addition, assays for GJ hemichannels demonstrated reduced plasmamembrane levels of connexin43 following PTX treatment. These resultssuggest that PTX-sensitive G proteins regulate connexin43 trafficking,and, as a result of inhibition with PTX, the number of plasma membranehemichannels available for GJ assembly is reduced.

     

Loss of transposase-DNA interaction may underlie the divergence of mariner family transposable elements and the ability of more than one mariner to occupy the same genome

Mariners are a large family of eukaryotic DNA-mediated transposable elements that move via a cut-and-paste mechanism. Several features of the evolutionary history of mariners are unusual. First, they appear to undergo horizontal transfer commonly between species on an evolutionary timescale. They can do this because they are able to transpose using only their own self-encoded transposase and not host-specific factors. One consequence of this phenomenon is that more than one kind of mariner can be present in the same genome. We hypothesized that two mariners occupying the same genome would not interact. We tested the limits of mariner interactions using an in vitro transposition system, purified mariner transposases, and DNAse I footprinting. Only mariner elements that were very closely related to each other (ca. 84% identity) cross-mobilized, and then inefficiently. Because of the dramatic suppression of transposition between closely related elements, we propose that to isolate elements functionally, only minor changes might be necessary between elements, in both inverted terminal repeat and amino acid sequence. We further propose a mechanism to explain mariner diversification based on this phenomenon.     

Mutagenesis of Neisseria meningitidis by in vitro transposition of Himar1 mariner

Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or PCR-amplified meningococcal DNA, which is subsequently reintroduced into N. meningitidis by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the Himar1 insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.     

Identification of connexin-43 interacting proteins

Connexin-43 (Cx43), the most ubiquitously expressed vertebrate gap junction protein, has been shown to interact directly with Zonula Occludens-1 (ZO-1). Although several potential functions have been proposed for the ZO-1/Cx43 interaction, the role that ZO-1 and other Cx43-interacting partners play in the regulation of Cx43 trafficking, assembly, gating and turnover are not well understood. We believed a thorough analysis and classification of other Cx43-interacting proteins might help us to understand and better test these roles. We approached this question by utilizing Tandem Mass Spectrometry (MS/MS) analysis to identify proteins from normal rat kidney whole cell lysates that could interact with the C-terminal region of Cx43. Comparison against protein sequence databases identified 19 probable protein matches, including kinases, phosphatases, membrane receptors, cell signaling molecules and scaffolding proteins. We have further characterized some of these interacting proteins, including Zonula Occludens-2 (ZO-2), via western blotting and "pull down" experiments. Further in vitro/in vivo analysis of these interacting proteins will help in our understanding of the global role of connexins in regulating development, cell metabolism and growth.     

CD134 as target for specific drug delivery to auto-aggressive CD4<Superscript>+</Superscript>T cells in adjuvant arthritis

T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4⁺ T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134⁺ T cells showed a specific proliferative response to the disease-associated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4⁺CD134⁺ T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'-fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134⁺ T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4⁺ T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development.     

Connexin 43 interacts with zona occludens-1 and -2 proteins in a cell cycle stage-specific manner

Gap junction channels play an important role in cell growth control, secretion and embryonic development. Gap junctional communication and channel assembly can be regulated by protein-protein interaction with kinases and phosphatases. We have utilized tandem mass spectrometry (MS/MS) sequence analysis as a screen to identify proteins from cell lysates that interact with the C-terminal cytoplasmic region of connexin 43 (Cx43). MS/MS analysis of tryptic fragments yielded several proteins including zona occludens-1 (ZO-1), a structural protein previously identified to interact with Cx43, and ZO-2, a potential novel interacting partner. We confirmed the interaction of ZO-2 with Cx43 by using a combination of fusion protein "pull down," co-immunoprecipitation, and co-localization experiments. We show that the C-terminal region of Cx43 is necessary for interaction with the PDZ2 domain of ZO-2. Far Western analysis revealed that ZO-2 can directly bind to Cx43 independent of other interacting partners. Immunofluorescence studies indicate that both ZO-1 and ZO-2 can co-localize with Cx43 within the plasma membrane at apparent gap junctional structures. We examined Cx43 interaction with ZO-1 and ZO-2 at different stages of the cell cycle and found that Cx43 had a strong preference for interaction with ZO-1 during G0, whereas ZO-2 interaction occurred approximately equally during G0 and S phases. Since essentially all of the Cx43 in G0 cells is assembled into Triton X-100-resistant junctions, Cx43-ZO-1 interaction may contribute to their stability.     

Establishment of a Genetically Marked Insect-Derived Symbiont in Multiple Host Plants

Alcaligenes xylosoxidans subsp. denitrificans, originally isolated from the cibarial region of the foregut of the glassy-winged sharpshooter (Homalodisca coagulata), was transformed using the Himar1 transposition system to express EGFP. Seedlings of six potential host plants were inoculated with transformed bacteria and 2 weeks later samples were taken 5 cm away and analyzed by quantitative real-time PCR using primers designed to amplify the gene insert. The largest colony of 3,591,427 cells/2 cm of A. xylosoxidans subsp. denitrificans was found in Citrus limon, with almost all plants testing positive in both trials. The amount of colonization decreased in the other plants tested in the following order: orange (Citrus sinensis sweet orange) > chrysanthemum (Chrysanthemum grandiflora cv. White Diamond) > periwinkle (Vinca rosea) > crepe myrtle (Lagerstroemia indica) > grapevine (Vitis vinifera cv. Chardonnay). The bacteriums preference for citrus paralleled the host insects preference for this same plant. Additional tests determined that A. xylosoxidans subsp. denitrificans thrives as a nonpathogenic, xylem-associated endophyte.