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Cacodylate Buffer (cacodylate + buffer)
Selected AbstractsMicroinjected neutrophils retain the ability to take up bacteriaJOURNAL OF ANATOMY, Issue 5 2002M. M. Bird It is now possible to microinject protein to probe specific biochemical pathways and/or cell functions in small cells such as human neutrophils (Bird et al. J.Anat.198, 2001). We have shown that these cells retain their ability to modify their F-actin cytoskeleton following the microinjection procedure. The principal task of neutrophils is to hunt and kill bacteria by responding to chemotactic gradients which cause them to extend actin rich pseudopodia in the direction of the highest concentration of these molecules. On reaching their target the neutrophils make tight contact with the bacteria and phagocytosis ensues. Here we address the question of whether or not the microinjected cells are still able to maintain their normal phagocytic activities. Human neutrophils maintained in culture for 20 mins were confronted with Staphylococcus aureus (1 × 104 cells/mL) for 5 min and then injected with rat IgG as an exogenous protein that also serves as a marker for injected cells. After 30 min the cells were fixed for fluorescence or confocal microscopy in 3.7% formaldehyde and permeabilised for 5 min (0.2% Triton X-100 in PBS). They were then incubated for 45 min in 2.5 µL FITC-anti rat IgG and 1 µL TRITC-phalloidin (to show the F-actin cytoskeleton), in 996.5 µL of PBS, washed 6 times in PBS and mounted on slides in 5 µL Mowiol containing a grain of antiquench. For TEM cells were fixed in 1.5% glutaraldehyde in cacodylate buffer for 3 min at room temperature and then washed in 0.2 m cacodylate buffer 6 times before incubation with 1 mm NiCl2 and SIGMA fast DAB peroxidase tablets for 30 min. The cells were postfixed in a 2% solution of osmium tetroxide for 30 min, dehydrated through a series of graded ethanols, and embedded and sectioned for TEM. By TEM the injected neutrophils were observed to have taken up bacteria into vacuoles of varying size. At the earliest stages of this process, prior to and immediately following the initial release of granular contents and the initiation of mechanisms to rapidly destroy bacteria, the bacteria fitted more tightly in the vacuoles than at later stages. Injected neutrophils commonly contained several bacteria; more than one bacterium was frequently located within a single vacuole of substantial size. Confocal laser microscopic observations confirmed that cells containing ingested bacteria also contained IgG. Thus injected cells not only survive the microinjection procedure but also retain their ability to take up bacteria and initiate the digestive process. [source] Simultaneous fixation using glutaraldehyde and osmium tetroxide or potassium ferricyanide-reduced osmium for the preservation of monogenean flatworms: An assessment for Merizocotyle icopaeMICROSCOPY RESEARCH AND TECHNIQUE, Issue 2 2004Bronwen Cribb Abstract Simultaneous fixation was investigated for a marine organism: the monogenean flatworm ectoparasite Merizocotyle icopae. Four protocols for primary fixation were compared: 3% glutaraldehyde alone in 0.1M cacodylate buffer for a minimum of 2 hours; 1% glutaraldehyde in combination with 1% osmium tetroxide, both in 0.1M cacodylate buffer, until tissues darkened (5,20 minutes); 1% glutaraldehyde in 0.1M cacodylate buffer in combination with 0.5% potassium ferricyanide-reduced osmium until tissues darkened (5,20 minutes); 1% glutaraldehyde in combination with 1% osmium tetroxide, both in 0.1M cacodylate buffer, for 30 minutes. The study confirms that the standard method for transmission electron microscopic fixation (first listed protocol) routinely applied to platyhelminths is optimal for ultrastructural preservation, but some simultaneous fixation methods (second and third listed protocols) are acceptable when rapid immobilization is needed. Scanning electron microscopic preparations may be improved using simultaneous primary fixation. Microsc. Res. Tech. 63:102,110, 2004. © 2004 Wiley-Liss, Inc. [source] Structure of SRP14 from the Schizosaccharomyces pombe signal recognition particleACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Mark A. Brooks The signal recognition particle (SRP) Alu domain has been implicated in translation elongation arrest in yeasts and mammals. Fission yeast SRP RNA is similar to that of mammals, but has a minimal Alu -domain RNA lacking two stem-loops. The mammalian Alu -domain proteins SRP9 and SRP14 bind their cognate Alu RNA as a heterodimer. However, in yeasts, notably Saccaromyces cerevisiae, SRP14 is thought to bind Alu RNA as a homodimer, the SRP9 protein being replaced by SRP21, the function of which is not yet clear. Structural characterization of the Schizosaccharomyces pombe Alu domain may thus help to identify the critical features required for elongation arrest. Here, the crystal structure of the SRP14 subunit of S. pombe SRP (SpSRP14) which crystallizes as a homodimer, is presented. Comparison of the SpSRP14 homodimer with the known structure of human SRP9/14 in complex with Alu RNA suggests that many of the protein,RNA contacts centred on the conserved U-turn motif are likely to be conserved in fission yeast. Initial attempts to solve the structure using traditional selenomethionine SAD labelling failed. However, two As atoms originating from the cacodylate buffer were found to make cysteine adducts and strongly contributed to the anomalous substructure. These adducts were highly radiation-sensitive and this property was exploited using the RIP (radiation-damage-induced phasing) method. The combination of SAD and RIP phases yielded an interpretable electron-density map. This example will be of general interest to crystallographers attempting de novo phasing from crystals grown in cacodylate buffer. [source] Crystallization and preliminary diffraction analysis of a ,-galactosidase from Trichoderma reeseiACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2009Mirko Maksimainen An extracellular ,-galactosidase from Trichoderma reesei was crystallized from sodium cacodylate buffer using polyethylene glycol (PEG) as a precipant. Crystals grown by homogenous streak-seeding belonged to space group P1, with unit-cell parameters a = 67.3, b = 69.1, c = 81.5,Å, , = 109.1, , = 97.3, , = 114.5°. The crystals diffracted to 1.8,Å resolution using a rotating-anode generator and to 1.2,Å resolution using a synchrotron source. On the basis of the Matthews coefficient (VM = 3.16,Å3,Da,1), one molecule is estimated to be present in the asymmetric unit. The aim of the determination of the crystal structure is to increase the understanding of this industrially significant enzyme. [source] | ||||||||||