Fluorescence microscopy visualization of halomucin, a secreted 927 kDa protein surrounding Haloquadratum walsbyi cells

At the time of its first publication, halomucin from Haloquadratum walsbyi strain HBSQ001 was the largest archaeal protein known (9159 aa). It has a predicted signal sequence, making it likely to be an extracellular or secreted protein. Best BLAST matches were found to be mammalian mucins that protect tissues to dehydration and chemical stress. It was hypothesized that halomucin participates in protection against desiccation by retaining water in a hull around the halophilic organisms that live at the limits of water activity. We visualized Halo quadratum cells by staining their intracellular polyhydroxybutyrate granules using Nile Blue. Halomucin was stained by immunofluorescence with antibodies generated against synthetic peptides derived from the halomucin amino acid sequence. Polyhydroxybutyrate stained cells were reconstructed in 3D which highlights not only the highly regular square shape but also the extreme flatness of Haloquadratum. Double-staining proves halomucin to be extracellular but to be only loosely associated to cells in agreement with its hypothesized function

The repertory of bone marrow progenitor cells associated with lymphogenic metastasis in patients with invasive carcinoma of no special type

The high mortality of patients with breast cancer is determined by metastatic disease. It is thought that the metastatic disease development associated with the repertory of bone marrow progenitor cells in breast cancer patients. In our study the correlation between the bone marrow progenitor cells presences in the tumor and blood of patients and the lymphogenic metastasis development was studied. The main clinical and pathological parameters of 24 patients with invasive breast carcinoma of non-specific type were analyzed. Endothelial progenitor cells, mesenchymal stem cells, macrophage precursors, hematopoietic progenitor cells were detected with specific antibodies against CD34, CD133, CD90, VEGFR1, CD11b, CD45, CD202 in the cell-rich fluid from frozen tumor. The amount of MCP-1 in the patients blood serum was assessed by enzymelinked immunosorbent assay (ELISA), at a wavelength of 450 nm. The cytokines concentration was calculated from the calibration plot. The program package Statistica 10.0. was used for statistical data processing. The high risk of lymphogenic metastasis in patients who didn't complete a neoadjuvant chemotherapy course was associated with the number of HPC, EPC and MSC in tumor and MCP-1 in blood

The problem of assessing the quality of health

This article analyzes the concept of "health" and the problem of assessing the quality of health

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system

Specific Age-Associated DNA Methylation Changes in Human Dermal Fibroblasts

Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (<23 years) and elderly donors (>60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner

Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks

Cells in culture undergo replicative senescence. In this study, we analyzed functional, genetic and epigenetic sequels of long-term culture in human mesenchymal stem cells (MSC). Already within early passages the fibroblastoid colonyforming unit (CFU-f) frequency and the differentiation potential of MSC declined significantly. Relevant chromosomal aberrations were not detected by karyotyping and SNP-microarrays. Subsequently, we have compared DNA-methylation profiles with the Infinium HumanMethylation27 Bead Array and the profiles differed markedly in MSC derived from adipose tissue and bone marrow. Notably, all MSC revealed highly consistent senescence-associated modifications at specific CpG sites. These DNA-methylation changes correlated with histone marks of previously published data sets, such as trimethylation of H3K9, H3K27 and EZH2 targets. Taken together, culture expansion of MSC has profound functional implications - these are hardly reflected by genomic instability but they are associated with highly reproducible DNA-methylation changes which correlate with repressive histone marks. Therefore replicative senescence seems to be epigenetically controlled

The PANDA GEM-based TPC Prototype

We report on the development of a GEM-based TPC prototype for the PANDA experiment. The design and requirements of this device will be illustrated, with particular emphasis on the properties of the recently tested GEM-detector, the characterization of the read-out electronics and the development of the tracking software that allows to evaluate the GEM-TPC data.Comment: submitted to NIMA 4 pages, 6 picture

Wnt5a induces ROR1 to associate with 14-3-3ζ for enhanced chemotaxis and proliferation of chronic lymphocytic leukemia cells.

Wnt5a can activate Rho GTPases in chronic lymphocytic leukemia (CLL) cells by inducing the recruitment of ARHGEF2 to ROR1. Mass spectrometry on immune precipitates of Wnt5a-activated ROR1 identified 14-3-3ζ, which was confirmed by co-immunoprecipitation. The capacity of Wnt5a to induce ROR1 to complex with 14-3-3ζ could be blocked in CLL cells by treatment with cirmtuzumab, a humanized mAb targeting ROR1. Silencing 14-3-3ζ via small interfering RNA impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance in vitro exchange activity of ARHGEF2 and (3) induce activation of RhoA and Rac1 in CLL cells. Furthermore, CRISPR/Cas9 deletion of 14-3-3ζ in ROR1-negative CLL cell-line MEC1, and in MEC1 cells transfected to express ROR1 (MEC1-ROR1), demonstrated that 14-3-3ζ was necessary for the growth/engraftment advantage of MEC1-ROR1 over MEC1 cells. We identified a binding motif (RSPS857SAS) in ROR1 for 14-3-3ζ. Site-directed mutagenesis of ROR1 demonstrated that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2 to ROR1, and activation of RhoA and Rac1. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1 signaling, leading to enhanced CLL migration and proliferation

PAK1 modulates a PPARγ/NF-κB cascade in intestinal inflammation

P21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases with both kinase and scaffolding activity. Here, we investigated the effects of inflammation on PAK1 signaling and its role in colitis-driven carcinogenesis. PAK1 and p-PAK1 (Thr423) were assessed by immunohistochemistry, immunofluorescence, and Western blot. C57BL6/J wildtype mice were treated with a single intraperitoneal TNFα injection. Small intestinal organoids from these mice and from PAK1-KO mice were cultured with TNFα. NF-κB and PPARγ were analyzed upon PAK1 overexpression and silencing for transcriptional/translational regulation. PAK1 expression and activation was increased on the luminal intestinal epithelial surface in inflammatory bowel disease and colitis-associated cancer. PAK1 was phosphorylated upon treatment with IFNγ, IL-1β, and TNFα. In vivo, mice administered with TNFα showed increased p-PAK1 in intestinal villi, which was associated with nuclear p65 and NF-κB activation. p65 nuclear translocation downstream of TNFα was strongly inhibited in PAK1-KO small intestinal organoids. PAK1 overexpression induced a PAK1–p65 interaction as visualized by co-immunoprecipitation, nuclear translocation, and increased NF-κB transactivation, all of which were impeded by kinase-dead PAK1. Moreover, PAK1 overexpression downregulated PPARγ and mesalamine recovered PPARγ through PAK1 inhibition. On the other hand PAK1 silencing inhibited NF-κB, which was recovered using BADGE, a PPARγ antagonist. Altogether these data demonstrate that PAK1 overexpression and activation in inflammation and colitis-associated cancer promote NF-κB activity via suppression of PPARγ in intestinal epithelial cells