L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

cDNAs coding for the HLA class II DR and DQ and chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded and chains of the DR3 or DR4 haplotypes, as well as the trans-encoded and chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR/DQ or DQ/DR) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most pan-DQ mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U07848. The name DQB1 ^* 0202 was officially assigned by the WHO Nomenclature Committee in April 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

Modulation of cell surface GABA(B) receptors by desensitization, trafficking and regulated degradation

Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.     

Fusing VE-Cadherin to α-Catenin Impairs Fetal Liver Hematopoiesis and Lymph but Not Blood Vessel Formation

We have recently shown that genetic replacement of VE-cadherin by a VE-cadherin–α-catenin fusion construct strongly impairs opening of endothelial cell contacts during leukocyte extravasation and induction of vascular permeability in adult mice. Here we show that this mutation leads to lethality at midgestation on a clean C57BL/6 background. Investigating the reasons for embryonic lethality, we observed a lack of fetal liver hematopoiesis and severe lymphedema but no detectable defects in blood vessel formation and remodeling. As for the hematopoiesis defect, VE-cadherin–α-catenin affected neither the generation of hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium nor their differentiation into multiple hematopoietic lineages. Instead, HSPCs accumulated in the fetal circulation, suggesting that their entry into the fetal liver was blocked. Edema formation was caused by disturbed lymphatic vessel development. Lymphatic progenitor cells of VE-cadherin–α-catenin-expressing embryos were able to leave the cardinal vein and migrate to the site of the first lymphatic vessel formation, yet subsequently, these cells failed to form large lumenized lymphatic vessels. Thus, stabilizing endothelial cell contacts by a covalent link between VE-cadherin and α-catenin affects recruitment of hematopoietic progenitors into the fetal liver and the development of lymph but not blood vessels.     

The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

An application programming interface for CellNetAnalyzer

CellNetAnalyzer (CNA) is a MATLAB toolbox providing computational methods for studying structure and function of metabolic and cellular signaling networks. In order to allow non-experts to use these methods easily, CNA provides GUI-based interactive network maps as a means of parameter input and result visualization. However, with the availability of high-throughput data, there is a need to make CNA's functionality also accessible in batch mode for automatic data processing. Furthermore, as some algorithms of CNA are of general relevance for network analysis it would be desirable if they could be called as sub-routines by other applications. For this purpose, we developed an API (application programming interface) for CNA allowing users (i) to access the content of network models in CNA, (ii) to use CNA's network analysis capabilities independent of the GUI, and (iii) to interact with the GUI to facilitate the development of graphical plugins.Here we describe the organization of network projects in CNA and the application of the new API functions to these projects. This includes the creation of network projects from scratch, loading and saving of projects and scenarios, and the application of the actual analysis methods. Furthermore, API functions for the import/export of metabolic models in SBML format and for accessing the GUI are described. Lastly, two example applications demonstrate the use and versatile applicability of CNA's API. CNA is freely available for academic use and can be downloaded from http://www.mpi-magdeburg.mpg.de/projects/cna/cna.html.     

GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)-related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β(2) integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β(2) integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.     

The structure of (μ3-sulfido)(μ3-p-tolyphosphido)tris(tricarbonyliron)(2FeFe)

Compounds of the type (μ₃-S)(μ₃-RP)Fe₃(CO)₉ have been prepared by the reaction of Fe₃(CO)₁₂ with [RP(S)S]₂ or RP(S)Cl₃. In this paper we report the synthesis of (μ₃-S)(μ₃-CH₃OC₆H₄P)Fe₃(CO)₉ using Lawesson's reagents, and the three dimensional structure of (μ₃-S)(μ₃-p-CH₃C₆H₄P)Fe₃(CO)₉. This material crystallizes in the space group P2₁/n with a = 8.558(2) Å, b = 9.022(2) Å, c = 27.506(6) Å, β = 95.40(2)°, Z = 4. The cluster is a nido structure as found commonly for (μ₃-X)(μ₃-Y)Fe₃(CO)₉ complexes.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.