Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.     

Mouse chromosome 15

Committee Members: R. Duncan and J. Todd.     

Cloning and expression of the essential gene for guanylate kinase from yeast.

Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the acceptor molecule GMP. Detailed analysis of the in vivo function of this enzyme has been limited by the lack of any genetic data. Using oligonucleotides based on amino acid sequence information of the yeast enzyme, the Saccharomyces cerevisiae gene, GUK1, was isolated and characterized. The gene is present in single copy and maps to chromosome IV. Insertional mutagenesis of the GUK1 locus caused recessive lethality, indicating that this enzyme is necessary for vegetative cell growth. Using inducible expression systems, guanylate kinase was produced in large amounts both in S. cerevisiae and in Escherichia coli.     

The influence of ants on patterns of colonization and establishment within a set of coexisting lycaenid butterflies in a south-east Asian tropical rain forest

In Peninsular Malaysia ten species of lycaenid butterflies use leaf flushes or inflorescences of the legume tree Saraca thaipingensis as larval hostplant. Resource partitioning among these species is regulated by a complex mixture of patterns of interaction with ants. Females of obligately myrmecophilous species lay their eggs exclusively on trees colonized by their specific host ants. On trees colonized by weaver ants, only specialist mutualists adapted to these territorial ants are able to survive, while larvae of other species are killed. The formicine ant Cladomyrma petalae, which inhabits hollow twigs of the myrmecophytic hostplant, likewise precludes oviposition by female butterflies. Lycaenid larvae confronted with this ant species never survive, but one concealed feeding species (Jamides caeruleus) escapes removal due to the cryptic life-habits of the larvae. Two facultative myrmecophiles associate in a mutualistic way with a wide and largely overlapping range of ant genera which forage at the extrafloral nectaries of leaf flushes. One species (Cheritra freja) is not myrmecophilous, but is tolerated by all but the most territorial ants. Ant-dependent hostplant selection and egg-clustering characterize the obligate mutualists, whereas facultative myrmecophiles and the non-myrmecophile distribute their eggs singly over appropriate hostplants. Signals mediating caterpillar-ant communication are highly specialized in one obligate myrmecophile (Drupadia theda), but rather unspecific in four other species tested. Altogether our observations indicate that colonization and establishment of lycaenid butterflies on S. thaipingensis trees are governed by specializations as well as opportunistic use of resources (ants and hostplant parts). Therefore, the diversity of this species assemblage is maintained by deterministic as well as stochastic factors.     

Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation.

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.     

Hyper-recombination in dam mutants of Escherichia coli K-12

Summary F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30–200 times higher than the isogenic dam ⁺ strain. A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant. The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers. It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.     

Immunohistochemical characterization of the thymic microenvironment

Summary The epithelial framework of the human thymus has been studied in parallel by immunohistochemical methods at the light- and electron-microscopic levels. Different monoclonal antibodies were used, reacting with components of the major histocompatibility complex, keratins, thymic hormones and other as yet antigenically undefined substances, which show specific immunoreactivities with human thymus epithelial cells.The electron-microscopic immunocytochemical observations clearly confirm microtopographical differences of epithelial cells not only between the thymic cortex and medulla, but also within the cortex itself. At least four subtypes of epithelial cells could be distinguished: 1) the cortical surface epithelium; 2) the main cortical epithelial cells and thymic nurse cells; 3) the medullary epithelial cells; and 4) the epithelial cells of Hassall's corpuscles.The various epithelial cell types of the thymus display several common features like tonofilaments, desmosomes and some surface antigens as demonstrated by anti-KiM3. In other respects, however, they differ from each other. The cortical subtype of thymic epithelial cells including the thymic nurse cells shows a distinct pattern of surface antigens reacting positively with antibodies against HLA-DR (anti-HLA-DR) and anti-21A62E. Electron-microscopic immunocytochemistry with these antibodies clearly reveals a surface labeling and a narrow contact to cortical thymocytes particularly in the peripheral cortical regions. An alternative staining pattern is realized by antibodies to some antigens associated with other subtypes of thymic epithelial cells. Medullary epithelial cells as well as the cortical surface epithelium react likewise positively with antibodies to special surface antigens (anti-Ep-1), to special epitopes of cytokeratin (anti-IV/82), and to thymic hormones (anti-FTS). The functional significance of distinct microenvironments within the thymus provided by different epithelial cells is discussed in view of the maturation of T-precursor cells.Glossary of Abbreviations Anti-X anti-X antibody - APUD-cells amine precursor uptake and decarboxylation (gastro-intestinal endocrine cells) - DAB diamino-benzidine - DMSO dimethyl sulfoxide - FTS facteur thymique sérique - HLA-A, B, C human leucocyte antigen, A, B, C-region related - HLA-DR human leucocyte antigen, D-region related - IDC interdigitating cell - MHC major histocompatibility gene complex - PBS phosphate-buffered saline - TNC thymic nurse cell This investigation was supported by grants from the Deutsche Forschungsgemeinschaft, and its Sonderforschungsbereich 111Fellow of the Alexander von Humbold-Stiftung, Institute of Pathology, University of Würzburg, Federal Republic of GermanyThe authors appreciate the contribution of human thymus tissue from Professor Alexander Bernhard, Abteilung kardiovasculäre Chirurgie der Universität Kiel; the gift of monoclonal antibodies from Dr. M.J.D. Anderson, Dr. M. Dardenne and Dr. H.J. Radzun; and the excellent technical assistence of Mrs. O.M. Bracker, Mrs. H. Hansen, Mrs. R. Köpke, Mrs. M. v. Kolszynski, Mrs. J. Quitzau, Mrs. H. Siebke, and Mrs. H. Waluk     

Association between genetic variants of the metabotropic glutamate receptor 3 (GRM3) and cognitive set shifting in healthy individuals

Set‐shifting and maintenance are complex cognitive processes, which are often impaired in schizophrenia. The genetic basis of these processes is poorly understood. We aimed to investigate the association between genetic variants of the metabotropic glutamate receptor 3 (GRM3) and cognitive set‐shifting in healthy individuals. The relationship between 14 selected single nucleotide polymorphisms (SNPs) of the GRM3 gene and cognitive set‐shifting as measured by perseverative errors using the modified card sorting test (MCST) was analysed in a sample of N = 98 young healthy individuals (mean age in years: 22.7 ± 0.19). Results show that SNP rs17676277 is related to the performance on the MCST. Subjects with the TT genotype showed significantly less perseverative errors as compared with the AA (P = 0.025) and AT (P = 0.0005) and combined AA/AT genotypes (P = 0.0005). Haplotype analyses suggest the involvement of various SNPs of the GRM3 gene in perseverative error processing in a dominant model of inheritance. The findings strongly suggest that the genetic variation (rs17676277 and three haplotypes) in the metabotropic GRM3 is related to cognitive set‐shifting in healthy individuals independent of working memory. However, because of a relatively small sample size for a genetic association study, the present results are tentative and require replication.