Evaluation of Factors Related to Growth of Rift Valley Fever Virus in Suspended Cell Cultures

The effect of several controlled variables on the peak titer and fold increase of Rift Valley fever virus grown in suspension culture on two variants of Earle's L cell, L-DR and L-MA clone 1-1, was studied. No significant amount of cell-associated virus was found at 24 hr, indicating a release of virus soon after its formation. Mild sonic treatment of the virus produced in serum-free medium increased the infective titer about 10x. This difference was not observed with virus produced in medium supplemented with serum. Peak titer was not affected by medium used during the infection period, by multiplicity of inoculum (MOI), or by initial cell concentration within the test range of 10(4) to 2 x 10(6) cell/ml. Cell strain employed influenced titer, because the L-DR cell did not produce virus efficiently at low MOI and low initial cell concentration. The time of peak titer and fold replication was dependent on MOI and initial cell concentration. Differences in virus propagation in monolayer and suspension systems are discussed.     

Membranes of Saccharomyces cerevisiae

A crude small particle pellet, obtained from postmitochondrial supernatant fractions of Saccharomyces cerevisiae, contains about half the ergosterol and phospholipid of crude cell homogenates. Most of the phospholipid of this pellet is in a “heavy” fraction which, with the aid of electron microscopy, shows membranous elements in addition to discrete particles. The “heavy” fraction, upon treatment with deoxycholate, can be freed of membranes, or, with ribonuclease treatment, ribosomes can be removed, leaving relatively clean membranes. The “heavy” fraction resembles the microsomes of animal cells, but contains considerably less lipids, including phospholipids, thus suggesting a less well-developed intracellular membrane system.     

Spectrophotometric Measurements of Phytochrome in vivo and Their Correlation with Photomorphogenic Responses of Phaseolus

Direct in vivo measurements of phytochrome have been made in Phaseolus vulgaris by 2-filter difference spectrophotometry (Ratiospect). All measurements were made at 730 versus 800 nm and it is assumed that the Δ (ΔOD) is directly proportional to the PFR concentration of phytochrome present. Dose response curves were determined for both physiological and spectrophotometric responses for red induction and far-red photoinactivation. For induction, saturation occurs at 100 mj/cm² and for inactivation at 30 mj/cm². The rate of hook opening and the physiological response measured 20 hours after induction are both shown to be directly proportional to the initial amount of PFR present spectrophotometrically. The sensitivity of the tissue correlates well with the absolute amount of phytochrome present, the inner portion of the hook having the maximum concentration of 0.042 Δ (ΔOD)/g fresh weight. If the total reversible phytochrome concentration is reduced by exposure to red light and allowing PFR to decay out of the system the remaining sensitivity of the tissue is shown to be directly correlated with the amount of PR remaining in the tissue. PFR disappears rapidly in the dark at 25°, and is not detectable after 6 hours. There is no indication that PFR reverts in the system to PR. At 4°, PFR does not disappear measurably up to 1 hour and is nearly totally reversible to PR.     

Resistance to H-2-restricted but not to allo-H2-specific graft and cytotoxic T lymphocyte responses in lymphoma mutant

The lymphoma mutant RMA-S escaped graft rejection after transplantation over a minor histocompatibility barrier, whereas it was rejected in H-2 allogeneic mice. The parental control line was rejected in both situations. The mutant, which had been selected against MHC class I molecules retained 5 to 10% of the wild-type H-2Db, Kb, and beta 2-microglobulin expression on the cell surface. It remained sensitive to allo-H-2b CTL in vitro, but was completely resistant to minor histocompatibility antigen-specific, H-2b-restricted CTL. It was equally resistant to other H-2b-restricted responses against internally derived Ag, such as tumor-specific CTL or a CTL clone specific for the influenza virus nucleoprotein. The results indicate a target cell defect that selectively abolishes the sensitivity to H-2-restricted CTL directed against internally processed Ag. This appears sufficient to shift the transplantation response over a minor histocompatibility Ag barrier from rejection to acceptance. There are two possible explanations for the results: 1) a block in the MHC class I-directed pathway for internal Ag processing, and 2) subthreshold H-2/Ag ligand density in relation to triggering requirements of restricted CTL. Regardless of the type of defect, the results demonstrate a difference between allo-H-2-specific and H-2-restricted CTL recognition at the level of the target cell.     

A rapid posttranslational myristylation of a 68-kD protein in D. discoideum

Cells incubated with [3H]myristate were shown to rapidly and specifically acylate a 68-kD protein, p68, in a developmentally-regulated manner. The fatty acid incorporated into p68 was identified as myristate, and is linked to the protein via an amide bond, apparently to an NH2-terminal glycine. The acylation of p68 in D. discoideum displays some unusual properties. Unexpectedly, myristylation of p68 is a posttranslational event and occurs in the presence of inhibitors of protein synthesis. Another unusual finding was that although p68 is a stable protein, the acyl moiety is removed with a half time of approximately 15 min.