Immunogenic and protective response in mice immunized with a purified, inactivated, Dengue-2 virus vaccine prototype made in fetal rhesus lung cells.

The feasibility of a purified, inactivated vaccine (PIV) against dengue type 2 (DEN-2) virus was explored. Dengue-2 virus strain 16681 was used for producing a monotypic PIV. Virus adapted to fetal rhesus lung (FRhL-2) cells was harvested from roller bottle culture supernatant fluids, concentrated, and purified on sucrose gradients. Analysis of purified virus preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed primarily envelope (E) and premembrane (prM) antigens. These preparations had a purity, estimated from silver-stained gels, of approximately 70%, and a yield, based on recovery of virus and viral antigen, of 10-20%. The purified virus was inactivated with 0.05% formalin at 22 degrees C, or alternatively, with 7 mRads from a 60Co source. Vaccinated mice developed high titers of anti-DEN-2 virus neutralizing antibody and were partially protected from virus challenge. These results warrant further testing and development of PIVs for the other DEN virus serotypes.

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil.

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [(3)H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.

Protein synthesis and degradation during expression of the temperature-sensitive defect in ts A1S9 mouse L-cells.

The involvement of altered protein metabolism in the expression of the temperature-sensitive (ts) pleiotropic phenotype of ts A1S9 cells was investigated. Cells are ts in growth and DNA replication. They undergo decondensation of their heterochromatin, interruptions of chromatin synthesis, and changes in cell size and morphology at the non-permissive temperature (npt) of 38.5 degrees C. Whereas the rates of incorporation of 3H-leucine, 35S-methionine, and 3H-fucose into proteins were unaffected at 38.5 degrees C, net protein accumulation was greatly reduced. This imbalance resulted from a rapid increase in the rate of protein degradation at the npt. Enhancement of protein degradation was detected within 2-4 hours after temperature upshift and constitutes the earliest metabolic alteration thus far observed during expression of the temperature-sensitive phenotype. The average half-life of proteins performed in ts A1S9 cells at 34 degrees C was decreased four-fold at the npt, and all major cytoplasmic proteins were affected equally. Enhanced protein degradation at the npt was shown to be sensitive to cycloheximide, ammonia, chloroquine, and vinblastine at concentrations that did not affect the basal protein degradation of normally cycling cells. Increased protein degradation at 38.5 degrees C did not involve an equivalent increase in total cellular protease activity. The data obtained are compatible with a model that suggests that temperature inactivation of the ts A1S9 gene product results in activation of a lysosome-mediated mechanism for the rapid degradation of cytoplasmic proteins.

Vectorial transport of proteins by membrane-bound ribosomes of nongrowing and growing Jerusalem artichoke tuber cells.

Both nongrowing (water-incubated) and growing (hormonally stimulated) Jerusalem artichoke tuber cells contain membrane-bound (mb) ribosomes. Using a rapid flotation procedure, a membrane fraction was prepared from both types of cells. This fraction was enriched in mb ribosomes, contained NADH cytochrome c reductase activity, had RNA:phospholipid and RNA:protein ratios similar to those reported for rough microsomes from animal tissues, and supported synthesis of preinitiated proteins in vitro. Using puromycin and detergent release, vectorial transport of labelled polypeptides was measured in the in vitro system. Of proteins made by mb ribosomes from nongrowing cells, on 12% remained associated with microsome membranes following chain termination. The comparable figure for proteins from mb ribosomes of growing tissue was 42%. The membrane-associated proteins were preferentially protected from protease digestion. Some possible reasons are suggested for the correlation between cell growth and the association of newly synthesized proteins with microsomes. The role of proteins synthesized by mb ribosomes but not vectorially transported, in both growing and nongrowing cells, is unknown.

Metabolism of free and membrane-bound ribosomes during aging of Jerusalem artichoke tuber slices.

Changes in ribosomes of artichoke (Helianthus tuberosus L.) tuber cells following excision and aging of tissue slices in water were studied using biochemical techniques. During the first 2 h of aging total rRNA dropped 28% and then remained constant for a subsequent 46 h. Since ribosome synthesis occurs through at least the first 24 h of aging, turnover of ribosomes must take place in this period. Cells of the dormant tuber gave essentially no membrane-bound (mb) ribosomes. On aging, the mb ribosome fraction rose and reached a maximum of 25% of total ribosomes at 24 h. Density gradient analysis showed that the ribosomes of dormant cells were present largely as monosomes. After 4 h aging a significant number of ribosomes in both free and mb populations sedimented as polysomes and the number of polysomes in both populations increased to a maximum at 24 h. The direct polysome analysis was confirmed by estimates of synthetically "active" ribosomes obtained using 0.8 M KCl to isolate monosomes carrying nascent polypeptides. This approach showed that while unaged cells had only 13% of total ribosomes active, on aging the active fraction rose to about 68% at 24 h. Both free and mb populations showed the same percentage of ribosomes active at all times studied. [(3)H]uridine showed significant incorporation into ribosomes during three periods studied; 2-4h, 12-14h, 22-24h. At the two latter periods the specific activity of the free ribosomes was greater than that of the mb ribosomes. Uridine was incorporated into both active and inactive ribosomes of both populations, judged by KCl fractionation, with the inactive fraction having greater specific activity in both cases. These differences in labelling possibly result from relatively slow mixing of different ribosome populations. Uptake of soluble [(3)H]uridine into the tissue increased 4-fold between 4 h and 14 h accounting at least in part for greater overall specific activity of ribosomes at later aging times.