Monolayer cultures of human epithelial and endothelial cells were used to study the association of latent transforming growth factor-beta 1 (TGF-beta 1) to extracellular matrices and its release and activation during matrix degradation. Human umbilical vein endothelial cells and embryonic lung fibroblasts produced relatively high levels of TGF-beta 1, its propeptide (beta 1-latency-associated protein), and latent TGF-beta-binding protein and incorporated latent TGF-beta 1 into their matrices as shown by immunoblotting. Amnion epithelial cells produced lower levels of these proteins. Confluent cultures of epithelial cells were exposed to matrix-degrading proteases and glycosidases. Mast cell chymase, leukocyte elastase, and plasmin efficiently released matrix-bound latent TGF-beta 1 complexes, while chondroitinase ABC and heparitinases were ineffective. The ability of the proteases to activate recombinant latent TGF-beta 1 was tested using growth inhibition assays and a novel sodium deoxycholate-polyacrylamide gel electrophoresis followed by immunoblotting. Sodium deoxycholate solubilized M(r) 25,000 TGF-beta 1 but did not dissociate high M(r) latent TGF-beta 1 complexes, allowing separation of these forms by polyacrylamide gel electrophoresis. Mast cell chymase and leukocyte elastase did not activate latent TGF-beta 1, suggesting that its release from matrix and activation are controlled by different mechanisms. The release of TGF-beta from the matrix by leukocyte and mast cell enzymes may contribute to the accumulation of connective tissue in inflammation.

STAT6 is a central mediator of IL‐4‐induced gene responses. STAT6‐mediated transcription is depend ent on the C‐terminal transcription activation domain (TAD), but the mechanisms by which STAT6 activates transcription are poorly understood. Here, we have identified the staphylococcal nuclease (SN)‐like domain and tudor domain containing protein p100 as a STAT6 TAD interacting protein. p100 was originally characterized as a transcriptional coactivator for Epstein–Barr virus nuclear antigen 2. STAT6 interacted with p100 in vitro and in vivo. The interaction was mediated by the TAD domain of STAT6 and the SN‐like domain of p100. p100 did not affect the immediate activation events of STAT6, but enhanced STAT6‐mediated transcriptional activation and the IL‐4‐induced Igϵ gene transcription in human B‐cell line. Finally, p100 associated with the large subunit of RNA polymerase II and was mediating interaction between STAT6 and RNA polymerase II. These findings identify p100 as a novel coactivator for STAT6 and suggest that p100 functions as a bridging factor between STAT6 and the basal transcription machinery.

N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrixbound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinaseactive fractions that bind to the cytosolic moiety of Nsyndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway. HB-GAM1 was initially isolated from neonatal rat brain as a neurite outgrowth-promoting protein, the expression of which in brain corresponds to the stage of rapid axonal growth (1). Molecular cloning of full-length cDNA identified a novel secretory sequence (2). The same cDNA sequence was reported for pleiotrophin, a protein suggested to be mitogen for fibroblastic cells (3, 4). The HB-GAM/pleiotrophin sequence shares approximately 50% homology with the midkine protein involved in retinoic acid-induced cell differentiation (5-7).The expression of HB-GAM in the axon pathways of the brain and in the basement membranes outside of brain (8 -9) and the neurite outgrowth-promoting property of HB-GAM in vitro (see Refs. 1 and 4) have suggested interaction of matrixassociated HB-GAM with a cell surface receptor. Furthermore, HB-GAM is expressed at the surface of developing muscle cells and is suggested to play a role in the development of nerve/ muscle contacts (10 -12). N-syndecan (syndecan-3) has recently been isolated from detergent extracts of perinatal rat brain as a receptor or coreceptor for HB-GAM using recombinant HB-GAM as an affinity matrix (13). N-syndecan is localized at the surface of neurites and their growth cones in rat primary neurons growing on HB-GAM-coated matrix in vitro (13). Furthermore, HB-GAM and N-syndecan are spatiotemporally co-expressed in developing rat brain (14).The cell surface N-syndecan interacts with HB-GAM through its heparan sulfate chains (15). This interaction is enhanced by assembly of the heparan sulfat...

Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies. STEM CELLS 2007;25: 197-202

As a source of transforming growth factor beta1 (TGF-beta1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF-beta1 have remained vague. We show here by means of RT-PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF-beta1 in their chymase 1-containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF-beta1, which is converted by chymase 1 into an active form recognized by the type II TGF-beta serine/threonine kinase receptor (TbetaRII). Thus, mast cells secrete active TGF-beta1 by a unique secretory mechanism in which latent TGF-beta1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF-beta1 specifically by chymase was verified using recombinant human latent TGF-beta1 and recombinant human chymase. In isolated TbetaRI- and TbetaRII-expressing peritoneal macrophages, the activated TGF-beta1 induces the expression of the plasminogen activator inhibitor 1 (PAI-1), whereas in the mast cells, the levels of TbetaRI, TbetaRII, and PAI-1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF-beta1 in peritoneal mast cells and of TbetaRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF-beta1 and by enhancing its response in target cells.

Background: Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.

Plant virus-encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall-associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein.These data suggest that pectin methylesterase is a host cell receptor involved in cell-to-cell movement of tobacco mosaic virus.z 1999 Federation of European Biochemical Societies.

In order to enable long-term operation of autonomous vehicles in industrial environments numerous challenges need to be addressed. A basic requirement for many applications is the creation and maintenance of consistent 3D world models. This article proposes a novel 3D spatial representation for online real-world mapping, building upon two known representations: normal distributions transform (NDT) maps and occupancy grid maps. The proposed normal distributions transform occupancy map (NDT-OM) combines the advantages of both representations; compactness of NDT maps and robustness of occupancy maps. One key contribution in this article is that we formulate an exact recursive updates for NDT-OMs. We show that the recursive update equations provide natural support for multi-resolution maps. Next, we describe a modification of the recursive update equations that allows adaptation in dynamic environments. As a second key contribution we introduce NDT-OMs and formulate the occupancy update equations that allow to build consistent maps in dynamic environments. The update of the occupancy values are based on an efficient probabilistic sensor model that is specially formulated for NDT-OMs. In several experiments with a total of 17 hours of data from a milk factory we demonstrate that NDT-OMs enable real-time performance in large-scale, long-term industrial setups.