Temporal aspects of O-glycosylation and cell surface expression of ascites sialoglycoprotein-1, the major cell surface sialomucin of 13762 mammary ascites tumor cells

We have investigated the biosynthesis and cell surface expression of the major cell surface sialomucin (ascites sialoglycoprotein-1 (ASGP-1] of 13762 rat mammary ascites tumor cells by pulse or pulse-chase metabolic labeling combined with precipitation with peanut agglutinin and alkaline borohydride elimination or proteolytic fragmentation. The minimum time for initial glycosylation was estimated from the time required for the protein to acquire the ability to bind to peanut agglutinin to be less than 5 min. Moreover, when cells were labeled with threonine for 5 min and the ASGP-1 isolated by peanut agglutinin precipitation, 3% of the labeled threonine could be converted to 2-aminobutyric acid by alkaline borohydride elimination of the carbohydrate, indicating that at least 3% of the threonines of ASGP-1 are O-glycosylated within 5 min of polypeptide synthesis. The minimum time between the final glycosylation reactions in the cell and appearance of ASGP-1 at the cell surface was determined by trypsinizing galactose- or glucosamine-labeled cells at timed intervals after labeling to occur within 5-10 min of labeling. Both labeled glucosamine and galactosamine appeared in ASGP-1 fragments within 5 min, but the amount of labeled galactosamine was less than the amount of labeled glucosamine until after 20 min, when the 1:1 equilibrium ratio was reached. The half-time for appearance of glucosamine-labeled ASGP-1 at the cell surface was found to be greater than 4 h. The minimum time required from synthesis of the ASGP-1 polypeptide to appearance at the cell surface was determined by leucine labeling and proteolysis to be 70-80 min. These combined studies suggest a continuum of O-linked oligosaccharide initiation events extending over most of the period of ASGP-1 biosynthesis and transit from the endoplasmic reticulum to the cell surface.     

Isolation and characterization of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell microvilli

A 58-kDa protein is found in microvilli and in actin-containing transmembrane complexes of 13762 ascites tumor cells with immobile surface receptors; it is absent from sublines with mobile receptors. 58-kDa protein has been proposed to stabilize microvilli and restrict receptor mobility by stabilizing membrane-microfilament interactions. Antibodies against 58-kDa protein were blot-purified from antisera of rabbits injected with crude transmembrane complex and were used to monitor purification of the protein. 58-kDa protein was extracted from EDTA/EGTA-stripped microvillar microfilament cores with 1 M NaCl. A single depolymerization-polymerization cycle of the microfilaments, followed by solubilization of 58-kDa protein in 1 M NaCl and chromatography on hydroxyapatite-Sephadex G-150, purified the protein to greater than 95% homogeneity. The native molecular weight and frictional coefficient indicated a monomeric, asymmetric structure. 58-kDa protein bound F-actin in pelleting assays and inhibited polymerization of pyrenyl-actin. It also bound phosphatidylserine, phosphatidylinositol, and phosphatidylcholine vesicles in pelleting studies. Immunoblot analyses of endogenously and exogenously proteolyzed microvilli and their membranes and microfilament cores showed specific membrane and microfilament binding fragments of 28-30 kDa. The microfilament- and phospholipid-binding properties of 58-kDa protein and the localization of its proteolysis products are consistent with its proposed role in stabilizing membrane-microfilament interactions in the ascites cell microvilli.     

Nonimmunodominant regions are effective as building blocks in a streptococcal fusion protein vaccine

Identification of antigens that elicit protective immunity is essential for effective vaccine development. We investigated the related surface proteins of group B Streptococcus, Rib and alpha, as potential vaccine candidates. Paradoxically, nonimmunodominant regions proved to be of particular interest as vaccine components. Mouse antibodies elicited by Rib and alpha were directed almost exclusively against the C-terminal repeats and not against the N-terminal regions. However, a fusion protein derived from the nonimmunodominant N-terminal regions of Rib and alpha was much more immunogenic than one derived from the repeats and was immunogenic even without adjuvant. Moreover, antibodies to the N-terminal fusion protein protected against infection and inhibited bacterial invasion of epithelial cells. Similarly, the N-terminal region of Streptococcus pyogenes M22 protein, which is targeted by opsonic antibodies, is nonimmunodominant. These data indicate that nonimmunodominant regions of bacterial antigens could be valuable for vaccine development.     

Unraveling the diversification and systematic puzzle of the highly polymorphic Psammobates tentorius (Bell, 1828) complex (Reptilia: Testudinidae) through phylogenetic analyses and species delimitation approaches

The high level of phenotypic diversity in southern African tent tortoises (Psammobates tentorius complex) has for decades prevented systematists from developing a stable taxonomy for the group. Here, we used a comprehensive DNA sequence dataset (mtDNA: Cytb, ND4, ND4 adjacent tRNA-His, and tRNA-Ser, 12S, 16S; and nDNA: PRLR gene) of 455 specimens, and the latest phylogenetic and species delimitation analytical procedures, to unravel the long-standing P. tentorius complex systematic puzzle. Our results for mtDNA and nDNA were incongruent, with the poorly supported nDNA phylogeny differentiating the three recognized subspecies, and showing potential hybridization in some regions. In contrast, the concatenated mtDNA phylogeny identified seven operational taxonomic units, with strong support. Clades 1, 4, 5, and 7 corresponded to tortoises identified as P. t. tentorius, clade 3 to P. t. trimeni, and clades 2 and 6 to P. t. verroxii. Our analyses showed conflicting topologies for the placement of C6 (P. t. verroxii north of the Orange River), with stronger support for it being sister to C2 + C3 than to the other clades. Clades 1, 2, and 6 had significantly higher genetic diversity than clades 3, 4, 5, and 7, perhaps because these clades inhabit substantially larger areas. The potential for future cladogenic radiations seems high in C1 and C6, particularly in C6 for which the within-clade diversification level was highest. Further research involving microsatellite DNA, phylogeographic evaluations, and morphological variation among clades is crucial for understanding the adaptive radiation of the P. tentorius complex and for modifying their taxonomy.     

Blockade of tissue factor-factor X binding attenuates sepsis-induced respiratory and renal failure

Tissue factor expression in sepsis activates coagulation in the lung, which potentiates inflammation and leads to fibrin deposition. We hypothesized that blockade of factor X binding to the tissue factor-factor VIIa complex would prevent sepsis-induced damage to the lungs and other organs. Acute lung injury was produced in 15 adult baboons primed with killed Escherichia coli [1 x 10(9) colony-forming units (CFU)/kg], and then 12 h later, they were given 1 x 10(10) CFU/kg live E. coli by infusion. Two hours after live E. coli, animals received antibiotics with or without monoclonal antibody to tissue factor intravenously to block tissue factor-factor X binding. The animals were monitored physiologically for 34 h before being killed and their tissue harvested. The antibody treatment attenuated abnormalities in gas exchange and lung compliance, preserved renal function, and prevented tissue neutrophil influx and bowel edema relative to antibiotics alone (all P < 0.05). It also attenuated fibrinogen depletion (P < 0.01) and decreased proinflammatory cytokines, e.g., IL-6 and -8 (P < 0.01), in systemic and alveolar compartments. Similar protective effects of the antibody on IL-6 and -8 expression and permeability were found in lipopolysaccharide-stimulated endothelial cells. Blockade of factor X binding to the tissue factor-factor VIIa complex attenuates lung and organ injuries in established E. coli sepsis by attenuating the neutrophilic response and inflammatory pathways.     

Molecular changes in cell surface membranes resulting from trypsinization of sarcoma 180 tumor cells.

Sarcoma-180 tumor cells in culture or grown as an ascites form in the CD-1 mouse have been subjected to mild trypsinization procedures in order to study morphological and molecular changes resulting from proteolysis. The cells attached to a substratum become rounded within 20 min. and most undergo cell division, but they do not detach from the substratum. Removal of trypsin permits the cells to go back to their original spindle shape over an 8-20 h period. Surface membranes were isolated from trypsinized ascites and cultured cells and subjected to dodecyl sulfate-acrylamide gel electrophoresis. Both cell types showed the same two kinds of changes in electrophoretic patterns. First, there was a loss of glycoproteins from both cell types even though they show different complements of cell surface glycoproteins. Second, there is a loss of high molecular weight polypeptides, which have previously been suggested to play a role in membrane stabilization and cell shape. These results further implicate these polypeptides in the control of cell morphology and offer circumstanital evidence for transmembrane interactions of surface glycoproteins with the high molecular weight polypeptides as a factor in controlling cell morphology.     

Towards proteome database of Francisella tularensis

The accessibility of the partial genome sequence of Francisella tularensis strain Schu 4 was the starting point for a comprehensive proteome analysis of the intracellular pathogen F. tularensis. The main goal of this study is identification of protein candidates of value for the development of diagnostics, therapeutics and vaccines. In this review, the current status of 2-DE F. tularensis database building, approaches used for identification of biologically important subsets of F. tularensis proteins, and functional and topological assignments of identified proteins using various prediction programs and database homology searches are presented.