Evidence that a protein-protein interaction 'hot spot' on heterotrimeric G protein betagamma subunits is used for recognition of a subclass of effectors.

To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.

Visible light-induced destabilization of endocytosed liposomes.

The potential biomedical utility of the photoinduced destabilization of liposomes depends in part on the use of green to near infrared light with its inherent therapeutic advantages. The polymerization of bilayers can be sensitized to green light by associating selected amphiphilic cyanine dyes, i.e. the cationic 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine (DiI), or the corresponding anionic disulfonated DiI (DiI-DS), with the lipid bilayer. The DiI sensitization of the polymerization of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-bis[10-(2', 4'-hexadienoyloxy)-decanoyl]-sn-glycero-3-phosphocholine liposomes caused liposome destabilization with release of encapsulated aqueous markers. In separate experiments, similar photosensitive liposomes were endocytosed by cultured HeLa cells. Exposure of the cells and liposomes to 550 nm light caused a net movement of the liposome-encapsulated 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) from low pH compartment(s) to higher pH compartment(s). This suggests that photolysis of DiI-labelled liposomes results in delivery of the contents of the endocytosed liposomes to the cytoplasm. The release of HPTS into the cytoplasm appears to require the photoactivated fusion of the labelled liposomes with the endosomal membrane. These studies aid in the design of visible light sensitive liposomes for the delivery of liposome-encapsulated reagents to the cytoplasm.

HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells.

We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.

Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction.

The human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site-directed mutagenesis. Asparagine residues of the consensus sequences (Asn-Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on immunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild type versus mutant receptors demonstrate that, of the 11 potential sites for N-linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, -488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface. Disruption of five glycosylation sites reduced cell surface expression by 50-90% depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction.

The bactericidal action of peroxides; an E.P.R. spin-trapping study.

E.P.R. spin trapping has been employed to study radical production during the bactericidal action of three peroxide compounds (peracetic acid, 4-percarboxy-N-isobutyltrimellitimide and magnesium monoperoxyphthalate) upon both Gram negative (Escherichia Coli) and Gram positive (Staphylococcus Aureus) bacteria. Use of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has allowed direct detection of both carbon-centred and hydroxyl radicals, which are produced at varying rates for the different bacteria/peracid systems studied. The inhibition of bactericidal action, by DMPO and two antioxidants, Vitamin C and Trolox C, indicates that radicals are the lethal species and evidence is presented which suggests that radical production is internal to the bacterial cell. Hydroxyl radicals are believed to be the lethal species. The effect of added iron chelators and haem protein inhibitors indicates that iron species and haem proteins in particular are involved. A marked variation is found in observed hydroxyl-radical adduct signals with both the nature and concentration of peracid. A strong inverse correlation is found between the concentration of the observed radical adduct signal and the relative strength of the peroxide as a bactericide; use of a stable nitroxide as a radical scavenger confirms that strong bactericides produce radicals at a much faster rate than weak bactericides. Plots of radical generation versus time are correlated with % bacterial kill, offering further evidence that hydroxyl radicals are the lethal species.

Emphysematous prostatic abscess: diagnosis and treatment.

We report an unusual case of an emphysematous prostatic abscess. Prostatic abscess is a difficult clinical diagnosis associated with lower urinary tract symptomatology and frequently diabetes mellitus. Computerized axial tomography and transrectal or transurethral ultrasonography can assist in making a specific diagnosis. Definitive treatment is complete surgical drainage, which is achieved by transurethral resection of the prostate. Wide spectrum, adjuvant antibiotic therapy should be given to assure coverage of anaerobic bacteria.

Iliocaval thrombi in pulmonary thromboembolic disease.

Rational management of patients with pulmonary thromboembolic disease should include assessment of the risk of additional emboli. Other authors have shown that the possibility of fatal pulmonary embolism is higher when the iliofemoral system contains thrombus, and it is recommended that vena caval interruption rather than simple anticoagulation is indicated. Additional factors governing the therapeutic choice should include the magnitude of the original embolic occlusion as well as the presence of antecedent cardiopulmonary disease. In these instances large thrombi in the iliocaval system should be regarded as potentially life threatening. A sequence of angiography beginning with right iliac and vena caval opacification, proceeding to pulmonary arteriography, and terminating with retrograde left iliac vein study provided information needed to individualize the therapeutic approach. Several case reports illustrate the spectrum of abnormalities and their therapeutic implications.