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51.
Summary Although providing superior histological preservation, Zenker-formol fixation has not been utilized in radioautographic experiments, since some component of the fixative (presumably mercury) desensitizes the emulsion and thus prevents the appearance of a radioautographic reaction. Attempts to identify and eliminate the effects of this desensitizing agent led to experiments with Zenker-formol fixed tissues from rats injected with 3H-leucine or 3H-thymidine. Radioautographs of such tissues contained a brown-black precipitate and occasionally a heavy background fog, but no normal radioautographic reaction. Moreover, when the radioautographs were exposed to light, the emulsion was transparent over the tissues, indicating that a tissue-bound component had completely desensitized the emulsion. The two components of Zenker's fluid-mercuric chloride and potassium dichromate-plus the mercurous chloride which forms as a precipitate in the course of fixation, were then tested for their individual effects on the radioautographic emulsion. It was found that mercuric chloride was primarily responsible for the desensitization of the emulsion. Merourous chloride, which formed the dark precipitate in radioautographs, produced the background fog. While potassium dichromate caused some desensitization of the emulsion, in normal histological processing where tissues had a lengthy post-fixation wash, this substance was for the most part washed out.A number of attempts were made to remove the mercury by treating the Zenker-fixed tissues with solutions of iodine, sodium thiosulfate and cysteine. It was found that the most intense radioautographic reaction was obtained with the following procedure: prior to embedding, tissue blocks were treated with 0.5% iodine solution in 70% alcohol for 17 hours. Optionally, sections of this material could be given a second treatment with 0.5% alcoholic iodine for 10 minutes. Although simple iodination eliminated the mercurous chloride precipitate and the background fog, there was still no radioautographic reaction. However, subsequent treatment of sections of this iodinated material in 5% aqueous sodium thiosulfate for 5 minutes yielded an adequate radioautographic reaction. Therefore, the simultaneous removal of both mercurous chloride precipitate and tissue bound mercury required iodination followed by sodium thiosulfate. Using this procedure, a method was described for preparing tissues for radioautography using Zenker-formol fixation and the periodic acid-Schiff staining technique.This work was supported by a grant from the Medical Research Council of Canada to Dr. C. P. Leblond. 相似文献
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Wittgens Andreas Kovacic Filip Müller Markus Michael Gerlitzki Melanie Santiago-Schübel Beatrix Hofmann Diana Tiso Till Blank Lars Mathias Henkel Marius Hausmann Rudolf Syldatk Christoph Wilhelm Susanne Rosenau Frank 《Applied microbiology and biotechnology》2017,101(7):2865-2878
Applied Microbiology and Biotechnology - The human pathogenic bacterium Pseudomonas aeruginosa produces rhamnolipids, glycolipids with functions for bacterial motility, biofilm formation, and... 相似文献
54.
Weigand JE Sanchez M Gunnesch EB Zeiher S Schroeder R Suess B 《RNA (New York, N.Y.)》2008,14(1):89-97
Riboswitches are genetic control elements that regulate gene expression in a small molecule-dependent way. We developed a two-stage strategy of in vitro selection followed by a genetic screen and identified several artificial small molecule-binding riboswitches that respond to the aminoglycoside neomycin. Structure-function relationships and structural probing revealed that they adopt the general neomycin-binding motif. They display no sequence similarities to in vitro selected neomycin aptamers but contain parts of the decoding site that is the binding site for neomycin on the ribosomal RNA. We propose a model of a composed binding pocket of an internal loop as primary docking site and a terminal flaplike loop structure fixing neomycin in a sandwich-like manner. Such binding pockets characterized by multiple contacts between ligand and RNA are described for both natural and engineered riboswitches. We anticipate that combination of in vitro selection and in vivo screening is a useful strategy to identify RNA molecules with a desired functionality. 相似文献
55.
Herndler-Brandstetter D Veel E Laschober GT Pfister G Brunner S Walcher S Parson W Lepperdinger G Grubeck-Loebenstein B 《Biological chemistry》2008,389(5):561-568
The age-related decline in immune system functions is responsible for the increased prevalence of infectious diseases and the low efficacy of vaccination in elderly individuals. In particular, the number of peripheral naive T-cells declines throughout life and they exhibit severe functional defects at advanced age. However, we have recently identified a non-regulatory CD8+CD45RO+ CD25+ T-cell subset that occurs in a subgroup of healthy elderly individuals, who still exhibit an intact humoral immune response following influenza vaccination. Here, we demonstrate that CD8+CD45RO+CD25+ T-cells share phenotypic and functional characteristics with naive CD8+CD45RA+CD28+ T-cells from young individuals, despite their expression of CD45RO. CD8+CD45RO+ CD25+ T-cells also have long telomeres and upon antigenic challenge, they efficiently expand in vitro and differentiate into functional effector cells. The expanded population also maintains a diverse T-cell receptor repertoire. In conclusion, CD8+CD45RO+CD25+ T-cells from elderly individuals compensate for the loss of functional naive T-cells and may therefore be used as a marker of immunological competence in old age. 相似文献
56.
Machado L Jarrett R Morgan S Murray P Hunter B Hamilton E Crocker J Thomas W Steven N Ismail T Chapman A Adams DH Lee SP 《Cancer immunology, immunotherapy : CII》2009,58(1):85-94
Circulating T lymphocytes enter a tissue if they express appropriate chemokine receptors and adhesion molecules to engage ligands presented at this site. To aid rational development of T cell-based therapies for Hodgkin's lymphoma (HL), we have assessed the expression and function of homing receptors on tumour-infiltrating T cells in HL and compared them with T cells from unaffected lymph nodes and colorectal cancer tissue. Chemokine receptors CXCR3, CXCR4 and CCR7 were expressed on a large proportion of T cells within HL tissue and mediated chemotaxis to purified chemokine. The corresponding ligands (CXCL10, CXCL12, CCL21) were expressed on the malignant cells and/or vascular endothelium. Adhesion molecules including CD62L were widely expressed on HL-derived T cells and their corresponding ligands were detected on vessels within the tumour. This homing phenotype was distinct from T cells isolated from colorectal cancer, but matched closely the phenotype of T cells from unaffected lymph nodes. Thus, T cell recruitment to HL resembles entry of na?ve/central memory T cells into normal lymph nodes. This has important implications for current approaches to treat HL using T cells activated and expanded in vitro that lack CCR7 and CD62L expression. 相似文献
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Steven Garvis Antje Munder Geneviève Ball Sophie de Bentzmann Lutz Wiehlmann Jonathan J. Ewbank Burkhard Tümmler Alain Filloux 《PLoS pathogens》2009,5(8)
Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity. 相似文献
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Karin Vitzthum Cristian Scutaru Lindy Musial-Bright David Quarcoo Tobias Welte Michael Spallek Beatrix Groneberg-Kloft 《PloS one》2010,5(6)