520 research outputs found
Virion Positions and Relationships of Lactococcal Temperate Bacteriophage TP901-1 Proteins
AbstractThe major proteins of phage TP901-1 virion were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and structural relations were determined using specific antibodies, obtained by affinity purification from a polyclonal serum. A 23-kDa protein was identified as the major tail protein, and a 31-kDa molecule as the major head protein, respectively. Labeling experiments with antibodies against two proteins, with molecular masses of 20 and 19 kDa, indicated that they were baseplate-related components. A 72-kDa protein was found to be part of a neck passage structure, which includes a collar. Evidence for the presence of attached whiskers was also obtained. T7 RNA polymerase-mediated expression of the two major proteins confirmed the gene location of the previously sequenced region of the phage genome. The relation to other lactococcal phages was determined by DNA hybridization and antibody probing, showing that despite low DNA similarity, TP901-1 NPS epitopes were detected in both related and unrelated small isometric-headed phages
Variations in local calcium signaling in adjacent cardiac myocytes of the intact mouse heart detected with two-dimensional confocal microscopy
Dyssynchronous local Ca release within individual cardiac myocytes has been
linked to cellular contractile dysfunction. Differences in Ca kinetics in
adjacent cells may also provide a substrate for inefficient contraction and
arrhythmias. In a new approach we quantify variation in local Ca transients
between adjacent myocytes in the whole heart. Langendorff-perfused mouse
hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize
cell membranes. A spinning disc confocal microscope with a fast camera allowed
us to record Ca signals within an area of 465 μm by 315 μm with an acquisition
speed of 55 fps. Images from multiple transients recorded at steady state were
registered to their time point in the cardiac cycle to restore averaged local
Ca transients with a higher temporal resolution. Local Ca transients within
and between adjacent myocytes were compared with regard to amplitude, time to
peak and decay at steady state stimulation (250 ms cycle length). Image
registration from multiple sequential Ca transients allowed reconstruction of
high temporal resolution (2.4 ± 1.3 ms) local CaT in 2D image sets (N = 4
hearts, n = 8 regions). During steady state stimulation, spatial Ca gradients
were homogeneous within cells in both directions and independent of distance
between measured points. Variation in CaT amplitudes was similar across the
short and the long side of neighboring cells. Variations in TAU and TTP were
similar in both directions. Isoproterenol enhanced the CaT but not the overall
pattern of spatial heterogeneities. Here we detected and analyzed local Ca
signals in intact mouse hearts with high temporal and spatial resolution,
taking into account 2D arrangement of the cells. We observed significant
differences in the variation of CaT amplitude along the long and short axis of
cardiac myocytes. Variations of Ca signals between neighboring cells may
contribute to the substrate of cardiac remodeling
Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake
Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the αv integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on αv integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn–expressing cells. Surprisingly, 30–50% of the endosomal contents were released into the cytosol of control and also K44A-dyn–expressing cells, and the number of fluid phase–positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection
Codetermination in the German Enterprise
We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome resequencing. Four of the mutants had different mutations in structural genes with no relation to the genetic switch. However all 8 mutants had a mutation in the cI repressor gene region. Three of these were located in the promoter and Shine-Dalgarno sequences and five in the N-terminal part of the encoded CI protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1
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