Biopsy coupled to quantitative immunofluorescence: a new method to study the human vascular endothelium.

Limited availability of endothelial tissue is a major constraint when investigating the cellular mechanisms of endothelial dysfunction in patients with metabolic and cardiovascular diseases. We propose a novel approach that combines collection of 200-1,000 endothelial cells from a superficial forearm vein or the radial artery, with reliable measurements of protein expression by quantitative immunofluorescence analysis. This method was validated against immunoblot analysis in cultured endothelial cells. Levels of vascular endothelial cell activation, oxidative stress, and nitric oxide synthase expression were measured and compared in five patients with severe chronic heart failure and in four healthy age-matched subjects. In summary, vascular endothelial biopsy coupled with measurement of protein expression by quantitative immunofluorescence analysis provides a novel approach to the study of the vascular endothelium in humans.     

Shaping up for shipping out: PLCgamma signaling of morphology changes in EGF-stimulated fibroblast migration

For effective migration, cells must establish an asymmetry in cell/substratum biophysical interactions permitting cellular protrusive and contractile motive forces to produce net cell body translocation; often this is superficially manifested as a polarized cell shape. This change is most easily noted for epithelial cells, which typically undergo a mesenchymal transition prior to rapid motility, and for hematopoietic cells, which must transition from non-adherent to adherent states. These two situations entail dramatic changes that also involve cell-cell contact and differentiation-related changes, and thus introduce confounding events and signals in defining control elements. Hence, a simpler biochemical and biophysical model system may be useful for gaining fundamental insights into the underlying mechanisms. Fortunately, even relatively "uniform" fibroblasts also undergo an initial shape change to commence locomotion. Investigators have recently begun to probe underlying signals that contribute to the reorganization of the actin cytoskeleton. We describe here a model for fibroblast shape changes involved in epidermal growth factor (EGF) stimulation of motility, focusing on signals through EGF receptor (EGFR) -mediated pathways influencing cytoskeletal organization and cell/substratum adhesion. We present new data addressing specifically phospholipase C-gamma (PLCgamma) pathway activation of actin-modifying proteins, including gelsolin, that contributes to these changes and promotes cell migration by increasing the fraction of cells in a motility-permissive morphology and the time spent in such a state.     

Electrophoretic mobilities and diffusion coefficients of hemoglobin at high pH.

Diffusion studies by photon correlation of scattered laser light confirm the dissociation of the tetrameric form of human carboxyhemoglobin to dimers above pH 10 and provide new estimates of the subunit dissociation equilibrium constants in this pH range. Electrophoretic light-scattering experiments under the same conditions reveal that the electrophoretic mobilities of tetramers and dimers are indistinguishable to within instrumental resolution (ca. 7% in these experiments). The data imply an increase of the electrical charge on the dimer of at least 2.8 to 4.4 net negative charges upon dissociation. Mechanisms for the accumulation of negative charge by the dimer upon dissociation of the tetramer are proposed.     

Structurally distinct recognition motifs in lymphotoxin-beta receptor and CD40 for tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.     

An improved method for the analysis of Cryptosporidium parvum oocysts by matrix-assisted laser desorption/ionization time of flight mass spectrometry

Cryptosporidium parvum oocysts were analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Sample preparation proved to be a crucial step in the acquisition of acceptable mass spectra. Oocysts of C. parvum and the matrix were mixed and held for at least 45 min to produce reproducible, representative mass spectra. Sporozoites were also excysted from oocysts, purified, and analyzed using MALDI-TOF MS. The mass spectra of the intact oocysts contained many of the same peaks found in the mass spectra of the sporozoites, suggesting that during analysis, the internal constituents, not just the oocyst wall, are ablated by the laser.