Complement resistance is essential for colonization of the digestive tract of Hirudo medicinalis by Aeromonas strains

From the crop of the medicinal leech, Hirudo medicinalis, only Aeromonas veronii bv. sobria can be cultured consistently. Serum-sensitive A. veronii mutants were unable to colonize H. medicinalis, indicating the importance of the mammalian complement system for this unusual simplicity. Complementation of one selected mutant restored its ability to colonize. Serum-sensitive mutants are the first mutant class with a colonization defect for this symbiosis.     

Repetitive versus monomeric antigen presentation: direct visualization of antibody affinity and specificity

The concept of presenting antigens in a repetitive array to obtain high titers of specific antibodies is increasingly applied by using surface-engineered viruses or bacterial envelopes as novel vaccines. A case for this concept was made 25 years ago, when producing high-titer antisera against ordered arrays of gp23, the major capsid protein of bacteriophage T4 (Aebi et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5514-5518). In view of the current interest in this concept we thought it useful to employ this system to directly visualize the dependence of antibody affinity and specificity on antigen presentation. We compared antibodies raised against T4 polyheads, a tubular variant of the bacteriophage T4 capsid, which have gp23 hexamers arranged in a crystalline lattice (gp23(repetitive)), with those raised against the hexameric gp23 subunits (gp23(monomeric)). The labeling patterns of Fab-fragments prepared from these antibodies when bound to polyheads were determined by electron microscopy and image enhancement. Anti-gp23(repetitive) bound in a monospecific, stoichiometric fashion to the gp23 units constituting the polyhead surface. In contrast, anti-gp23(monomeric) decorated the polyhead surface randomly and with a 40-fold lower occupancy. These results concur with the difference in titers established by ELISA for the antisera against the repetitively displayed form of antigen (anti-gp23(repetitive)) and the randomly presented antigen (gp23(monomeric)), and they constitute a compelling visual documentation of the concept of repetitive antigen presentation to elicite a serotype-like immune response.     

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.     

Design,synthesis, and structure-activity relationship of a new class of amidinophenylurea-based factor VIIa inhibitors

Selective inhibition of coagulation factor VIIa has recently gained attraction as interesting approach towards antithrombotic treatment. Using parallel synthesis supported by structure-based design and X-ray crystallography, we were able to identify a novel series of amidinophenylurea derivatives with remarkable affinity for factor VIIa. The most potent compound displays a K(i) value of 23 nM for factor VIIa.     

Reproduction of spined loach, Cobitis taenia, (Cypriniformes; Cobitidae) under laboratory conditions

A simple and reliable method is described for breeding spined loach, Cobitis taenia Linnaeus, 1758, under laboratory conditions. Three 40 L aquaria were each provided with a thick tuft of moss placed on top of a gauze-covered plastic box to serve as a spawning site. Ovoposition always occurred in the most densely available vegetation. The nonadhesive eggs fell through the gauze into the box. The spined loach is a fractional spawner releasing eggs in 14–18 batches at intervals of 2–23 (median 6) days. The total number of eggs per female was 2905–4282 during the reproductive season of 101–120 days. Yolks measured 1.14 ± 0.07 mm and eggs were 2.54 ± 0.22 mm in diameter. Total larval length was 5.03 ± 0.34 mm at hatching and 6.77 ± 0.34 mm at the beginning of exogenous feeding. The method allowed the rearing of numerous young for conservation measures and experimental investigations.     

UniqTag: Content-Derived Unique and Stable Identifiers for Gene Annotation

When working on an ongoing genome sequencing and assembly project, it is rather inconvenient when gene identifiers change from one build of the assembly to the next. The gene labelling system described here, UniqTag, addresses this common challenge. UniqTag assigns a unique identifier to each gene that is a representative k-mer, a string of length k, selected from the sequence of that gene. Unlike serial numbers, these identifiers are stable between different assemblies and annotations of the same data without requiring that previous annotations be lifted over by sequence alignment. We assign UniqTag identifiers to ten builds of the Ensembl human genome spanning eight years to demonstrate this stability. The implementation of UniqTag in Ruby and an R package are available at https://github.com/sjackman/uniqtag sjackman/uniqtag. The R package is also available from CRAN: install.packages ("uniqtag"). Supplementary material and code to reproduce it is available at https://github.com/sjackman/uniqtag-paper.     

Uncoupling of bait‐protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP‐1 and the PKA Cβ1 subunit

An expression‐uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP‐bait protein from the target cells. Here, the TAP‐tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue‐specific binding partners of the protein kinase A catalytic subunit Cβ1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor‐dependent interaction of Cβ1 with the cell cycle and apoptosis regulatory protein‐1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell‐ and tissue‐specific protein complexes.     

Interaction of hypochlorous acid and myeloperoxidase with phosphatidylserine in the presence of ammonium ions

The close association of the heme enzyme myeloperoxidase to phosphatidylserine epitopes on the surface of non-vital polymorphonuclear leukocytes (PMNs) and other apoptotic cells at inflammatory sites favours modifications of this phospholipid by myeloperoxidase products. As detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, ammonium ions inhibit in a concentration-dependent manner the hypochlorous acid-mediated formation of aldehyde and nitrile products from 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS). Concomitantly, the formation of monochloramine (NH₂Cl) raises with increasing NH₄⁺ concentrations. A transchlorination from monochlorinated O-phospho-l-serine to NH₄⁺ with the formation of NH₂Cl occurs only when extraordinary high NH₄⁺ concentrations are applied. Due to the low rate of 0.044 M^− 1 s^− 1 for this process, a transhalogenation reaction from transient chlorinated intermediates of the serine moiety to NH₄⁺ can be ruled out as an important process contributing to the HOCl-mediated formation of NH₂Cl. A significant formation of NH₂Cl by myeloperoxidase interacting with DPPS in the presence of ammonium ions takes only place at acidic pH values around 5, a scenario that may occur in phagosomes of macrophages after the uptake of apoptotic PMNs.