Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff?

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at 482VANTSTQTM downward arrowGPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.     

Transfection of a reporter plasmid into cultured cells by sonoporation in vitro

Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasound in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection. Ten percent of Albunex, a gas-body-based ultrasound contrast agent, was added to ensure cavitation nucleation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. Uptake of large fluorescent dextran molecules by some cells was observed for spatial peak pressure amplitudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure. Significant lysis occurred for 0.2 MPa, and increased rapidly for exposures above the apparent cavitation threshold (using the H2O2 production test) of about 0.4 MPa spatial peak pressure amplitude. In the DNA transfection tests, 20 micrograms/mL luciferase reporter plasmid was added to the suspension during exposure, and cells were assayed for proliferation ability and luciferase gene expression 2 days after exposure. Cell proliferation was greatly reduced above the cavitation threshold. Luciferase production was significant for 0.20-MPa exposure, and reached 0.33 ng per 10(6) cells at 0.8-MPa exposure. The luciferase production was great for cells exposed in medium supplemented with serum than for cells exposed in serum-free medium. Cells harvested for exposure either in the log phase or in the stationary phase of culture gave similar proliferation and transfection results. The effects essentially disappeared when the Albunex was omitted from the suspension and the tube was not rotated. Thus, sonoporation by ultrasonic cavitation in the rotating tube system yields plasmid transfection with subsequent transient gene expression.     

An evaluation of operator preference of diamond burs in coronal tooth preparation

Three postgraduate prosthodontic students served as clinicians/evaluators in a study rating their preferences for three different diamond cutting instruments from three manufacturers. Each evaluator prepared the axial walls of complete veneer crowns on extracted molar teeth and then ranked their preference of the instruments. To prepare nine teeth, each of the three instruments was used in random order and without knowledge of the specific manufacturer. The methodology for analyzing the evaluators' preferences and the results are discussed.     

A diminished role for hydrogen bonds in antifreeze protein binding to ice

The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP1) in winter flounder is a 37-amino-acid-long, alanine-rich, alpha-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {2021} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice.     

Effects of TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic mouse urinary tract

Lipoprotein(a) [Lp(a)] biogenesis was examined in primary cultures of hepatocytes isolated from mice transgenic for both human apolipoprotein(a) [apo(a)] and human apoB. Steady-state and pulse-chase labeling experiments demonstrated that newly synthesized human apo(a) had a prolonged residence time (approximately 60 min) in the endoplasmic reticulum (ER) before maturation and secretion. Apo(a) was inefficiently secreted by the hepatocytes and a large portion of the protein was retained and degraded intracellularly. Apo(a) exhibited a prolonged and complex folding pathway in the ER, which included incorporation of apo(a) into high molecular weight, disulfide-linked aggregates. These folding characteristics could account for long ER residence time and inefficient secretion of apo(a). Mature apo(a) bound via its kringle domains to the hepatocyte cell surface before appearing in the culture medium. Apo(a) could be released from the cell surface by apoB-containing lipoproteins. These studies are consistent with a model in which the efficiency of post-translational processing of apo(a) strongly influences human plasma Lp(a) levels, and suggest that cell surface assembly may be one pathway of human Lp(a) production in vivo. Transgenic mouse hepatocytes thus provide a valuable model system with which to study factors regulating human Lp(a) biogenesis.     

Leichte Turbolader-Verdichterräder aus Aluminium- und Magnesium-Feinguß

Light turbocharger compressor wheels from aluminium and magnesium investment casting Within the scope of a team work between Kühnle, Kopp & Kausch AG, Volkswagen AG, Sterling International Technologies Ltd. and the Fraunhofer Institute for Strength of Structures under Operational Conditions LBF compressor wheels of aluminium and magnesium investment casting were developed and tested. Strength and fatigue tests, overspeed and field tests as well as metallographic checks were made in addition to the finite element calculations to optimize the stress under centrifugal force in an appropriate way for the materials involved. The results and the knowledge gathered are to clarify whether magnesium alloys are an appropriate replacement for the proven standard aluminium alloy for turbochargers.     

Diffusion and transfer of antibody proteins from a sugar-based hydrogel

Diffusion of antibody protein from hydrogel films and hydrogel encapsulated in a microcapillary was studied. Thin hydrogel films were formed by crosslinking 6-acryloyl-B-O-methylgalactoside with N,N'-methylene-bis-acrylamide and the diffusive transport of monoclonal antimouse IgG-FITC into and out of the hydrate films was measured. Diffusion coefficients in 2 and 4% crosslinked hydrogel films were measured. The measured diffusion constants determined for IgG in both the 2 and 4% hydrogel films were comparable to the free diffusion of IgG in bulk water (Dmean approximately 10(-7) cm2/s). In addition, 2% crosslinked hydrogels were prepared in a capillary tube and the transport of antimouse IgG-FITC into and out of the hydrated hydrogel was measured. Kinetic analysis indicated that the protein transport through the capillary hydrogel was faster than would be expected for a simple diffusion process. Finally, by utilizing the diffusion of antibody from the capillary hydrogel, transfer of antibody to a silica surface was demonstrated. A capillary hydrogel loaded with antimouse IgG-FITC was used to transfer the protein to a silica surface forming a 30-micron spot of antibody, which was imaged using fluorescence microscopy. These results may lead to the development of a nonlithographic method of patterning antibodies on surfaces for use in integrated microimmunosensors.