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.     

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.     

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.     

Alanine and glutamine kinetics at rest and during exercise in humans

PURPOSE: The purpose of this study was to quantify both alanine and glutamine kinetics during exercise of moderate intensity to determine the sum total of alanine and glutamine flux. METHODS: Tracer methods were used to quantify alanine and glutamine rates of appearance (Ra) in plasma at rest and during 180 min of approximately 45% VO2max treadmill exercise in six normal volunteers (25 +/- 2 yr, 68 +/- 2.5 kg, VO2max 43 +/- 2.4 mL.min-1.kg-1; means +/- SE). Bolus injections (N = 3) or primed-constant infusions (N = 3) of 2H5-glutamine and 3-13C-alanine were given at rest on 1 d and 10-15 min after the onset of exercise on a separate day less than 2 wk later. Plasma enrichment decay curves and plateau enrichments were used to estimate alanine and glutamine kinetics. RESULTS: Whereas alanine Ra increased significantly from rest to exercise (5.72 +/- 0.31 vs 13.5 +/- 1.9 mumol.min-1.kg-1, respectively; P < 0.01), glutamine Ra was not significantly altered by exercise (6.11 +/- 0.44 and 6.40 +/- 0.69 mumol.min-1.kg-1 at rest and during exercise, respectively). The total of alanine and glutamine flux increased from 17.93 +/- 0.88 to 25.98 +/- 3.04 (P < 0.05). CONCLUSIONS: Since most muscle amino-N is released as alanine and glutamine, these findings provide strong evidence that amino-N delivery from muscle to the liver is increased during exercise. In addition, it appears that alanine, rather than glutamine, is the predominant N carrier involved in the transfer of N from muscle to the liver during moderate intensity exercise.     

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.