Trypanosoma cruzi: cell charge distribution

Diferenças na carga celular entre populações de epimastigotas e de tripomastigotas foram comparadas nas cepas Y, CL e Colombiana do T. cruzi. As populações de tripomastigotas mostraram-se mais homogêneas quanto à carga celular do que os epimastigotas. Esta maior homogeneidade de cargas não foi decorrente da seleção de sub-populações de tripomastigotas por ação do sistema imunológico do hospedeiro, mas talvez esta se deva à capa superficial formada por componentes sangüíneos dos hospedeiros.Differences in cell charge between epimastigote and trypomastigote populations were compared in Y, Cl and Colombiana strains of T. cruzi. Trypomastigote populations were more homogenous in relation to cell charge than epimastigotes. This homogeneity of cell charge was not the result of the selection of trypomastigote sub-populations by the host immunosystem, but may be the result of a surface coat formed by host blood components

Thin films of Type 1 collagen for cell by cell analysis of morphology and tenascin-C promoter activity

BACKGROUND: The use of highly reproducible and spatiallyhomogeneous thin film matrices permits automated microscopy and quantitative determination of the response of hundreds of cells in a population. Using thin films of extracellular matrix proteins, we have quantified, on a cell-by-cell basis, phenotypic parameters of cells on different extracellular matrices. We have quantitatively examined the relationship between fibroblast morphology and activation of the promoter for the extracellular matrix protein tenascin-C using a tenascin-C promoter-based GFP reporter construct. RESULTS: We find that when considering the average response from the population of cells, cell area correlates with tenascin-C promoter activity as has been previously suggested; however cell-by-cell analysis suggests that cell area and promoter activity are not tightly correlated within individual cells. CONCLUSION: This study demonstrates how quantitative cell-by-cell analysis, facilitated by the use of thin films of extracellular matrix proteins, can provide insight into the relationship between phenotypic parameters

Daily jaw muscle activity in freely moving rats measured with radio-telemetry.

The jaw muscle activity of rats has been investigated for specific tasks. However, the daily jaw muscle use remains unclear. The purpose of the present study was to examine daily jaw muscle activity, and its variability over time, in the rat (n = 12) by the use of radio-telemetry. A telemetric device was implanted for the continuous recording of masseter muscle and digastric muscle activity. Daily muscle use was characterized by calculating the total time that each muscle was active (duty time), the number of bursts, and the average length of bursts. All parameters were estimated for activities exceeding various levels (5-90%) of the day's peak activity. Daily muscle use remained constant for 4 wk. At the low-activity level, the duty time and burst number of the digastric muscle were significantly (P < 0.01) higher than those of the masseter muscle, whereas the opposite was true at the high-activity level (P < 0.05). No significant intermuscular correlation was observed between the number of bursts of the masseter and digastric muscles, but the interindividual variation of both muscles changed, depending on the level of activation. These findings suggest that the masseter muscle and the digastric muscle show a differential active pattern, depending on the activity level. © 2007 The Authors. Journal compilation 2007 Eur J Oral Sci

Quantifying myosin light chain phosphorylation in single adherent cells with automated fluorescence microscopy

<p>Abstract</p> <p>Background</p> <p>In anchorage dependent cells, myosin generated contractile forces affect events closely associated with adhesion such as the formation of stress fibers and focal adhesions, and temporally distal events such as entry of the cell into S-phase. As occurs in many signaling pathways, a phosphorylation reaction (in this case, phosphorylation of myosin light chain) is directly responsible for cell response. Western blotting has been useful in measuring intracellular phosphorylation events, but cells are lysed in the process of sample preparation for western blotting, and spatial information such as morphology, localization of the phosphorylated species, and the distribution of individual cell responses across the population is lost. We report here a reliable automated microscopy method for quantitative measurement of myosin light chain phosphorylation in adherent cells. This method allows us to concurrently examine cell morphology, cell-cell contact, and myosin light chain diphosphorylation in vascular smooth muscle cells.</p> <p>Results</p> <p>Paraformaldehyde fixation and Triton X-100 permeabilization preserved cell morphology and myosin light chain phosphorylation better than the alternative fixation/permeabilization methods tested. We utilized automated microscopy methods to acquire three color images, determine cell spread area, and quantify the intensity of staining within each cell with anti-phospho-MLC antibody. Our results indicate that A10 rat aortic smooth muscle cells exhibit a re producible non-Gaussian distribution of MLC phosphorylation across a population of unsynchronized genetically identical cells. Adding an inhibitor of Rho kinase, Y27632, or plating cells on a low density of fibronectin, reduced phospho-myosin light chain signal as expected. On the other hand, adding calyculin A, an activator of contractility, increased myosin light chain phosphorylation. The IC₅₀for myosin light chain phosphorylation using Y27632 was determined to be 2.1 ± 0.6 micrometers. We observed a positive linear relationship between cell area and myosin light chain diphosphorylation, which is consistent with what has been reported in the literature using other methods.</p> <p>Conclusion</p> <p>Our results show that using proper specimen fixation techniques and background subtraction methods, imaging cytometry can be used to reliably measure relative myosin light chain phosphorylation in individual adherent cells. Importantly, the ability to make this measurement in adherent cells allows for simultaneous measurement of and correlation with other parameters of cellular topography such as morphology and cell-cell proximity. This assay has potential application in screening for drug development.</p