The ADAM10 prodomain is a specific inhibitor of ADAM10 proteolytic activity and inhibits cellular shedding events

ADAM10 is a disintegrin metalloproteinase that processes amyloid precursor protein and ErbB ligands and is involved in the shedding of many type I and type II single membrane-spanning proteins. Like tumor necrosis factor-alpha-converting enzyme (TACE or ADAM17), ADAM10 is expressed as a zymogen, and removal of the prodomain results in its activation. Here we report that the recombinant mouse ADAM10 prodomain, purified from Escherichia coli, is a potent competitive inhibitor of the human ADAM10 catalytic/disintegrin domain, with a K(i) of 48 nM. Moreover, the mouse ADAM10 prodomain is a selective inhibitor as it only weakly inhibits other ADAM family proteinases in the micromolar range and does not inhibit members of the matrix metalloproteinase family under similar conditions. Mouse prodomains of TACE and ADAM8 do not inhibit their respective enzymes, indicating that ADAM10 inhibition by its prodomain is unique. In cell-based assays we show that the ADAM10 prodomain inhibits betacellulin shedding, demonstrating that it could be of potential use as a therapeutic agent to treat cancer.     

Antigen-independent immune responses after dendritic cell vaccination

The ability of cultured, antigen-loaded dendritic cells (DCs) to induce antigen-specific T cell immunity in vivo has previously been demonstrated and confirmed. Immune monitoring naturally focuses on immunity against vaccine antigens and may thus ignore other effects of DC vaccination. Here we therefore focused on antigen-independent responses induced by DC vaccination of renal cell carcinoma patients. In addition to the anticipated response against the vaccine antigen KLH, vaccination with CD83⁺ monocyte-derived DCs resulted in a strong increase in the ex vivo proliferative and cytokine responses of PBMCs stimulated with LPS or BCG. In addition, LPS strongly enhanced the KLH-induced proliferative and cytokine response of PBMCs. Moreover, proliferative and cytokine responses of PBMCs stimulated with the homeostatic cytokines IL-7 and IL-15 were also clearly enhanced after DC vaccination. In contrast to LPS induced proliferation, which is well known to depend on monocytes, IL-7 induced proliferation was substantially enhanced after monocyte depletion indicating that monocytes limit IL-7 induced lymphocyte expansion. Our data indicate that DC vaccination leads to an increase in the ex vivo responsiveness of patient PBMCs consistent with a DC vaccination induced enhancement of T cell memory. Our findings also suggest that incorporation of bacterial components and homeostatic cytokines into immunotherapy protocols may be useful in order to enhance the efficacy of DC vaccination and that monocytes may limit DC vaccination induced immunity. Supported by a grant to Martin Thurnher from the kompetenzzentrum medizin tirol (kmt), a center of excellence.     

Bee venom secretory phospholipase A2 and phosphatidylinositol-homologues cooperatively disrupt membrane integrity,abrogate signal transduction and inhibit proliferation of renal cancer cells

Bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P2) act synergistically to induce cell death in tumour cells of various origins with concomitant stimulation of the immune system. Here, we investigated the mechanisms involved in such actions and examined structural requirements of PtdIns-homologues to inhibit tumour cells in combination with bv-sPLA2. Renal cancer cells were treated with bv-sPLA2 alone or in combination with PtdIns-homologues. Inhibitory effects on [³H] thymidine incorporation and intracellular signal transduction pathways were tested. Reaction products generated by bv-sPLA2 interaction with PtdIns(3,4)P2 were identified by mass spectrometry. Among the tested PtdIns-homologues those with a phosphate esterified to position 3 of the inositol head group, were most efficient in cooperating with bv-sPLA2 to block tumour cell proliferation. Growth inhibition induced by the combined action of bv-sPLA2 with either PtdIns(3,4)bisphosphate or PtdIns(3,4,5)trisphosphate were synergistic and accompanied by potent cell lysis. In contrast, PtdIns, which lacked the phosphate group at position 3, failed to promote synergistic growth inhibition. The combined administration of PtdIns(3,4)P2 and bv-sPLA2 abrogated signal transduction mediated by extracellular signal regulated kinase 1 and 2 and prevented transduction of survival signals mediated by protein kinase B. Surface expression of the epidermal growth factor (EGF)-receptor was reduced after PtdIns(3,4)P2-bv-sPLA2 administration and associated with a blockade of EGF-induced signalling. In addition, mass spectroscopy revealed that bv-sPLA2 cleaves PtdIns(3,4)P2 to generate lyso-PtdIns(3,4)P2. In conclusion, we suggest that the cytotoxic activity mediated by PtdIns(3,4)P2 and bv-sPLA2 is due to cell death that results from disruption of membrane integrity, abrogation of signal transduction and the generation of cytotoxic lyso-PtdIns(3,4)P2. This work was supported by a grant to MT of the kompetenzzentrum medizin tirol (kmt), a centre of excellence.     

A colorimetric-based amplification system for proteinases including MMP2 and ADAM8

We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.     

A boy with classical Rubinstein-Taybi syndrome but no detectable mutation in the CREBBP and EP300 genes

Rubinstein-Taybi syndrome (RTS) is a rare autosomal dominant genetic disorder and is characterized by mental retardation, distinctive facial features, broad and often angulated thumbs and great toes. We report on a 7 year old boy with classical Rubinstein-Taybi syndrome. His facial and clinical features were very typical, including broad thumbs with radial angulation and broad great toes. Rigorous genetic analysis of the CREBBP and EP300 genes using DNA sequencing and multiple ligation-dependent probe amplification (MLPA) revealed no causative mutation in this boy, only a hitherto unreported but paternally inherited heterozygous sequence alteration, c.506 1+9C>T in IVS 30-31, which most likely represents a normal variant (NetGene 2 splice prediction software). We question if this boy could have a hitherto undetectable mutation type.     

GeneReporter--sequence-based document retrieval and annotation

GeneReporter is a web tool that reports functional information and relevant literature on a protein-coding sequence of interest. Its purpose is to support both manual genome annotation and document retrieval. PubMed references corresponding to a sequence are detected by the extraction of query words from UniProt entries of homologous sequences. Data on protein families, domains, potential cofactors, structure, function, cellular localization, metabolic contribution and corresponding DNA binding sites complement the information on a given gene product of interest. Availability and implementation: GeneReporter is available at http://www.genereporter.tu-bs.de. The web site integrates databases and analysis tools as SOAP-based web services from the EBI (European Bioinformatics Institute) and NCBI (National Center for Biotechnology Information).     

Lethal phenotype of mice carrying a Sept11 null mutation

Septins are cytoskeletal GTP-binding proteins involved in processes characterized by active membrane movement, such as cytokinesis, vesicle trafficking and exocytosis. Most septins are expressed ubiquitously, however, some septins accumulate in particular tissues. The ubiquitous SEPT11 also shows high expression levels in the central nervous system and in platelets. Here, SEPT11 is involved in vesicle trafficking and may play a role in synaptic connectivity. Interestingly, mice that harbor a homozygous Sept11 null mutation, die in utero. From day 11.5 post coitum onwards, development of homozygous embryos seems to be retarded and the embryos from day 13.5 onwards were dead.     

Cytokine profile of a human bone marrow cell culture under the influence of UHMW-PE wear particles]

There is considerable evidence that orthopaedic wear debris plays a crucial role in the pathology of aseptic loosening of joint prostheses. The purpose of the present study was to evaluate the influence of ultra-high-molecular-weight polyethylene (UHMW-PE) on the cytokine response in a modified in vitro model. UHMW-PE particles (psi < 7.5 microm) were suspended in soluble collagen type I and subsequently solidified in different concentrations (105,106 and 107 particles per well) on the bottom of the wells. Human bone marrow cells in a concentration of 3 x 106 cells per well were seeded on the collagen-particle substrata and maintained for up to 12 days. The cytokine response (IL-1_, IL-6 and TNF-_) of the cells to the particles were examined by ELISA compared to cells on control collagen surfaces without any particles. Assays for viability using LDH activity were done immediately. Light and scanning microscopic evaluation revealed that the UHMWPE particles, which have built large conglomerates (psi7.5_m), were mainly surrounded by the cells and less phagocytosed. The results of the cytokine release revealed significant differences in interleukin (IL)6, tumor necrosis factor (TNF)- _ and IL-1beta. The cell viability was not affected by the UHMW-PE particles. The results demonstrate that the particle induced cytokine response by UHMW-PE is mainly by the release of Interleukin 6 and TNF- _. Moreover the results confirm that the present method is useful to evaluate the in vitro effects of UHMW-PE wear particles with direct particle cell contact.     

Increased expression of bradykinin type-1 receptors in endothelium of intramyocardial coronary vessels in human failing hearts

In experimental animals, bradykinin type-1 receptors (BK-1Rs) are induced during inflammation and ischemia, and, by exerting either cardioprotective or cardiotoxic effects, they may contribute to the pathogenesis of heart failure. Nothing is known about the expression of BK-1Rs in human heart failure. Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n = 13), due to idiopathic dilated cardiomyopathy (IDC; n = 7) or to coronary heart disease (CHD; n = 6), and from normal hearts (n = 6). The expression of BK-1Rs was analyzed by means of competitive RT-PCR, Western blot analysis, and immunohistochemistry. Expression of BK-1R mRNA was increased in both IDC (2.8-fold) and CHD (2.1-fold) hearts compared with normal hearts. The observed changes were verified at the protein level. Expression of BK-1Rs in failing hearts localized to the endothelium of intramyocardial coronary vessels and correlated with an increased expression of TNF-alpha in the vessel wall. Treatment of human coronary artery endothelial cells with TNF-alpha increases their BK-1R expression. These novel results show that BK-1Rs are induced in the endothelium of intramyocardial coronary vessels in failing human hearts and so may participate in the pathogenesis of heart failure.