Extracts Of Untreated Cells Research Articles

Disaggregation and reconstitution of oligomeric complexes of simian virus 40 large T-antigen.

Biochemical properties of multiple species of simian virus 40 (SV40) large T-antigen produced in SV40-infected monkey cells were investigated by zonal sedimentation centrifugation of radiolabelled cell extracts on sucrose gradients. Two major subpopulations of T-antigen detected by immunoprecipitation and gel electrophoresis could be distinctly separated: low molecular weight forms ranging approximately between 5S and 10S and higher oligomeric forms at about 16S to 23S. Removal of divalent cations by chelating agents such as EDTA disassembled higher oligomers into low molecular weight forms (5S to 10S). Adding divalent cations in excess of the EDTA concentration reassembled the higher oligomeric forms, showing a sedimentation behaviour like T-antigen in untreated cell extracts. Our results are compatible with the hypothesis that divalent cation binding properties of T-antigen participate in the natural pathway of assembling multiple oligomeric species.

Cell cycle arrest as a basis of enhancement of 1-β- d-arabinofuranosylcytosine anabolism in human lymphoblastoid RPMI 6410 cells cultured with 3-deazauridine

The antiproliferative effect of 1-β- D-arabinofuranosylcytosine (araC) on RPMI 6410 cells in culture was potentiated in the presence of 3-deazauridine (DU). When the culture medium contained DU, proliferation ceased and cells did not progress in the replication cycle beyond early S phase. When cells from such DU-treated cultures were transferred to DU-free medium containing Cyd, a rapid, partially synchronous resumption of DNA synthesis occurred. Under these circumstances, the activity of dCyd kinase, assessed using araC as substrate, was enhanced in unfractionated extracts of DU-treated cells relative to extracts of untreated cells. It is suggested that the presence of DU in culture medium stopped the progression of cells through the replication cycle, arresting them in an araC-sensitive portion of S phase, and that the enhanced ability of DU-treated cells to anabolize dCyd and araC derived, at least in part, from the accumulation of cells in S phase, a portion of the cell cycle in which dCyd kinase is elevated.

Characteristics of Extracts from Interferon-treated HeLa Cells: Presence of a Protein Kinase and Endoribonuclease Activated by Double-stranded RNA and of an Inhibitor of mRNA Methylation

SUMMARY Extracts from mouse interferon-treated mouse cells (Ehrlich ascites tumour and L929) were reported earlier to differ in several characteristics from extracts of untreated cells. We report now that the effect of treatment with human interferon of cells of a human line (HeLa S3) is similarly manifested in the cell extract. A comparison of extracts from interferon-treated HeLa S3 cells (S30 int ) with that from untreated cells (S30c) revealed: (a) an impairment in S30 int of the methylation of (unmethylated) reovirus mRNAs by the cellular and virus enzymes; (b) a faster degradation of reovirus mRNAs in S30 int but only in reaction mixtures supplemented with double-stranded RNA and ATP; (c) an increased phosphorylation by ATP of one (or two) proteins in S30 int but only in reaction mixtures supplemented with double-stranded RNA and (d) a more pronounced inhibition of translation by double-stranded RNA in S30 int . No such effects were observed in extracts prepared from HeLa cells treated with mouse interferon.

A protein synthesizing system from interferon-treated cells that discriminates between cellular and viral messenger RNAs

The effect of treating Krebs II ascites tumor cells with interferon on the ability of extracts prepared from them to catalyze the translation of a variety of messenger RNAs was investigated. The following results were obtained: (1) Extracts of untreated cells translated endogenous mRNA, as well as exogenously added synthetic mRNA [poly(U)], cellular mRNA (Krebs cell and L cell), and viral mRNA (reovirus and vaccinia virus). By contrast, extracts of Krebs cells treated in the ascitic form with interferon translated endogenous mRNA, exogenously added cellular mRNA and poly(U) as efficiently as extracts of untreated cells, but they translated viral mRNAs very poorly (less than 10% as efficiently as extracts of untreated cells). Thus, the ability to discriminate between cellular and viral mRNAs that is characteristic of whole cells was also exhibited by these cell-free extracts. (2) Virus infection was not required for the development of the interferon-induced inhibition of viral mRNA translation. (3) Mixing experiments indicated that the inability of extracts of interferon-treated cells to catalyze the translation of viral mRNAs was due to the presence of an inhibitory factor (s) rather than to the absence of a required factor (s). (4) The inhibitory factor (s) was associated with ribosomes, and could be dissociated from them by washing with KC1. (5) Polyacrylamide gel electrophoresis revealed the presence of a 48,000 dalton polypeptide in the 0.3–0.6 M KC1 wash fraction of ribosomes prepared from interferon-treated cells that was not detectable in the corresponding wash of ribosomes prepared from untreated cells. This fraction inhibited the translation of viral mRNAs in cell-free extracts of untreated cells more than any other salt wash fraction. These results suggest that the antiviral activity of interferon is mediated, at least in part, by a ribosome-associated polypeptide that permits discrimination between cellular and viral mRNAs.

Serologic Studies on North American Blastomycosis

Summary and Conclusions A soluble antigen, obtained by sonic vibration of washed yeast-phase cells of Blastomyces dermatitidis, was used to test for complement-fixing antibodies in 165 sera, including 69 samples from patients with proved cases of blastomycosis and 15 from patients with a clinical diagnosis or suspected diagnosis of other fungus infections. The antigen, although failing to give positive results in 29% of cases of proved blastomycosis, proved to be as satisfactory as the yeast-phase antigens previously employed, and had the distinct advantages of solubility and ease of storage. The soluble materials from sonic-treated washed yeast-phase cells of Histoplasma capsulatum also gave positive complement fixation tests with sera from patients with proved blastomycosis. The supernatant fluid from the protein-free saline suspensions of untreated yeast-phase cells was shown to contain an antigen which fixed guinea pig complement with sera from immunized rabbits but not with sera from patients with blastomycosis. This supernatant fluid, and a polysaccharide precipitated from it, were capable of sensitizing sheep's cells, rendering them agglutinable by sera from patients with blastomycosis. There was no correlation between the titers obtained by the hemagglutination technique and those obtained by the complement fixation tests in sera of patients with blastomycosis, but there was a definite relationship between the titers obtained in both tests and the prognoses of the patients. The data obtained on a limited number of mice suggested that the injection of small amounts of carbohydrate material, precipitated from the saline extract of the untreated cells, stimulates the development of a limited degree of resistance to heavy challenge doses of living B. dermatitidis. Skin tests on guinea pigs infected with B. dermatitidis and Histoplasma capsulatum using extracts of untreated cells and treated cells of both fungi indicated that the former substances produced more specific reactions and that the latter antigens were less specific. The above findings and their implications in the interpretation of the immunologic reactions obtained in blastomycosis and other systemic fungus infections are discussed.